Crispr/cas fusion proteins and systems

ABSTRACT

Engineered Cas9 systems are disclosed herein.

RELATED APPLICATIONS

The present application is a Divisional Application of U.S. applicationSer. No. 17/172,734, filed February 10, 2021, which is a DivisionalApplication of U.S. application Ser. No. 16/790,399, filed February 13,2020, U.S. Pat. No. 10,947,517, which claims the benefit of priority ofU.S. Provisional Application No. 62/806,708, filed February 15, 2019,the entirety of each of which is incorporated herein by reference.

FIELD

The present disclosure relates to engineered Cas9 systems, nucleic acidsencoding said systems, and methods of using said systems for genomemodification.

BACKGROUND

Many different types of peptide linkers have been tested to fuse GFP toCas9, but typically result in lower activity of the underlying Cas9.

SUMMARY OF THE DISCLOSURE

Among the various aspects of the present disclosure include engineeredCas9 systems.

Other aspects and features of the disclosure are detailed bellow.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows that the Cas9 fusion proteins disclosed herein each retainthe editing activity parallel to the level of SpCas9 protein.

FIG. 2A and FIG. 2B show that the editing efficiencies of the Cas9fusion proteins disclosed herein were several-fold higher than that ofthe commercial proteins in all targets.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Apr. 27, 2023, isnamed P19027USDIV2_SEQLISTING_ASFILED.xml and is 119,483 bytes in size.

DETAILED DESCRIPTION

Fusion of accessory proteins to CRISPR proteins creates a wide range ofopportunities to localize various protein functionalities to definedlocations within cells. Among other things, peptide linkers which enablefusion of heterologous proteins to CRISPR proteins in ways that preserveCRISPR functionality are disclosed.

(I) Engineered Cas9 Systems

One aspect of the present disclosure provides engineered Cas9 proteinsand systems. For example, Cas9-marker fusion proteins are disclosed. Insome aspects, systems include engineered Cas9 proteins and engineeredguide RNAs, wherein each engineered guide RNA is designed to complexwith a specific engineered Cas9 protein. These engineered Cas9 systemsdo not occur naturally.

(a) Engineered Cas9 Proteins

Cas9 protein is the single effector protein in Type II CRISPR systems,which are present in various bacteria. The engineered Cas9 proteindisclosed herein can be from Acaryochloris sp., Acetohalobium sp.,Acidaminococcus sp., Acidithiobacillus sp., Acidothermus sp.,Akkermansia sp., Alicyclobacillus sp., Allochromatium sp., Ammonifexsp., Anabaena sp., Arthrospira sp., Bacillus sp., Bifidobacterium sp.,Burkholderiales sp., Caldicelulosiruptor sp., Campylobacter sp.,Candidatus sp., Clostridium sp., Corynebacterium sp., Crocosphaera sp.,Cyanothece sp., Exiguobacterium sp., Fibrobacter sp., Finegoldia sp.,Francisella sp., Ktedonobacter sp., Lachnospiraceae sp., Lactobacillussp., Listeria sp., Lyngbya sp., Marinobacter sp., Methanohalobium sp.,Microscilla sp., Microcoleus sp., Microcystis sp., Mycoplasma sp.,Natranaerobius sp., Neisseria sp., Nitratifractor sp., Nitrosococcussp., Nocardiopsis sp., Nodularia sp., Nostoc sp., Oenococcus sp.,Oscillatoria sp., Parasutterella sp., Pasteurella sp., Parvibaculum sp.,Pelotomaculum sp., Petrotoga sp., Polaromonas sp., Prevotella sp.,Pseudoalteromonas sp., Ralstonia sp., Rhodospirillum sp., Staphylococcussp., Streptococcus sp., Streptomyces sp., Streptosporangium sp.,Synechococcus sp., Thermosipho sp., Treponema sp., Verrucomicrobia sp.,and Wolinella sp.

Exemplary species that the Cas9 protein or other components may be fromor derived from include Acaryochloris spp. (e.g., Acaryochloris marina),Acetohalobium spp. (e.g., Acetohalobium arabaticum), Acidaminococcusspp., Acidithiobacillus spp. (e.g., Acidithiobacillus caldus,Acidithiobacillus ferrooxidans), Acidothermus spp., Akkermansia spp.,Alicyclobacillus spp. (e.g., Alicyclobacillus acidocaldarius),Allochromatium spp. (e.g., Allochromatium vinosum), Ammonifex spp.(e.g., Ammonifex degensii), Anabaena spp. (e.g., Anabaena variabilis),Arthrospira spp. (e.g., Arthrospira maxima, Arthrospira platensis),Bacillus spp. (e.g., Bacillus pseudomycoides, Bacillusselenitireducens), Bifidobacterium spp., Burkholderiales spp. (e.g.,Burkholderiales bacterium), Caldicelulosiruptor spp. (e.g.,Caldicelulosiruptor becscii), Campylobacter spp. (e.g., Campylobacterjejuni, Campylobacter lari), Candidatus spp., (e.g., Candidatusdesulforudis), Clostridium spp. (e.g., Clostridium botulinum,Clostridium difficile), Corynebacterium spp. (e.g., Corynebacteriumdiphtheria), Crocosphaera spp. (e.g., Crocosphaera watsonii), Cyanothecespp., Deltaproteobacterium spp., Exiguobacterium spp. (e.g.,Exiguobacterium sibiricum), (Fibrobacter spp. (e.g., Fibrobactersuccinogene), Finegoldia spp. (e.g., Finegoldia magna), Francisella spp.(e.g., Francisella novicida), Gammaproteobacterium, Ktedonobacter spp.(e.g., Ktedonobacter racemifer), Lachnospiraceae spp., Lactobacillusspp. (e.g., Lactobacillus buchneri, Lactobacillus delbrueckii,Lactobacillus gasseri, Lactobacillus salivarius), Listeria spp. (e.g.,Listeria innocua), Leptotrichia spp., Lyngbya spp., Marinobacter spp.,Methanohalobium spp. (e.g., Methanohalobium evestigatum), Microcoleusspp. (e.g., Microcoleus chthonoplastes), Microscilla spp. (e.g.,Microscilla marina), Microcystis spp. (e.g., Microcystis aeruginosa),Mycoplasma spp., Natranaerobius spp. (e.g., Natranaerobiusthermophilus), Neisseria spp. (e.g., Neisseria cinerea, Neisseriameningitidis), Nitratifractor spp., Nitrosococcus spp. (e.g.,Nitrosococcus halophilus, Nitrosococcus watsoni), Nocardiopsis spp.(e.g., Nocardiopsis dassonvillei), Nodularia spp. (e.g., Nodulariaspumigena), Nostoc spp., Oenococcus spp., Oscillatoria spp.,Parasutterella spp., Parvibaculum spp. (e.g., Parvibaculumlavamentivorans), Pasteurella spp. (e.g., Pasteurella multocida),Pelotomaculum spp., (e.g., Pelotomaculum thermopropionicum), Petrotogaspp. (e.g., Petrotoga mobilis), Planctomyces spp., Polaromonas spp.(e.g., Polaromonas naphthalenivorans), Prevotella spp.,Pseudoalteromonas spp. (e.g., Pseudoalteromonas haloplanktis), Ralstoniaspp., Ruminococcus spp., Rhodospirillum spp. (e.g., Rhodospirillumrubrum), Staphylococcus spp. (e.g., Staphylococcus aureus),Streptococcus spp. (e.g., Streptococcus pasteurianus, Streptococcuspyogenes, Streptococcus thermophilus), Sutterella spp. (e.g., Sutterellawadsworthensis), Streptomyces spp. (e.g., Streptomycespristinaespiralis, Streptomyces viridochromogenes, Streptomycesviridochromogenes), Streptosporangium spp. (e.g., Streptosporangiumroseum, Streptosporangium roseum), Synechococcus spp., Thermosipho spp.(e.g., Thermosipho africanus), Treponema spp. (e.g., Treponemadenticola), and Verrucomicrobia spp., Wolinella spp. (e.g., Wolinellasuccinogenes), and/or species delineated in bioinformatic surveys ofgenomic databases such as those disclosed in Makarova, Kira S., et al.“An updated evolutionary classification of CRISPR-Cas systems.” NatureReviews Microbiology 13.11 (2015): 722 and Koonin, Eugene V., Kira S.Makarova, and Feng Zhang. “Diversity, classification and evolution ofCRISPR-Cas systems.” Current opinion in microbiology 37 (2017): 67-78,each of which is hereby incorporated by reference herein in theirentirety.

In some embodiments, the engineered Cas9 protein may be fromStreptococcus pyogenes. In some embodiments, the engineered Cas9 proteinmay be from Streptococcus thermophilus. In some embodiments, theengineered Cas9 protein may be from Neisseria meningitidis. In someembodiments, the engineered Cas9 protein may be from Staphylococcusaureus. In some embodiments, the engineered Cas9 protein may be fromCampylobacter jejuni.

Wild-type Cas9 proteins comprise two nuclease domains, i.e., RuvC andHNH domains, each of which cleaves one strand of a double-strandedsequence. Cas9 proteins also comprise REC domains that interact with theguide RNA (e.g., REC1, REC2) or the RNA/DNA heteroduplex (e.g., REC3),and a domain that interacts with the protospacer-adjacent motif (PAM)(i.e., PAM-interacting domain).

The Cas9 protein can be engineered to comprise one or more modifications(i.e., a substitution of at least one amino acid, a deletion of at leastone amino acid, an insertion of at least one amino acid) such that theCas9 protein has altered activity, specificity, and/or stability.

For example, Cas9 protein can be engineered by one or more mutationsand/or deletions to inactivate one or both of the nuclease domains.Inactivation of one nuclease domain generates a Cas9 protein thatcleaves one strand of a double-stranded sequence (i.e., a Cas9 nickase).The RuvC domain can be inactivated by mutations such as D10A, D8A,E762A, and/or D986A, and the HNH domain can be inactivated by mutationssuch as H840A, H559A, N854A, N856A, and/or N863A (with reference to thenumbering system of Streptococcus pyogenes Cas9, SpyCas9). Inactivationof both nuclease domains generates a Cas9 protein having no cleavageactivity (i.e., a catalytically inactive or dead Cas9).

The Cas9 protein can also be engineered by one or more amino acidsubstitutions, deletions, and/or insertions to have improved targetingspecificity, improved fidelity, altered PAM specificity, decreasedoff-target effects, and/or increased stability. Non-limiting examples ofone or more mutations that improve targeting specificity, improvefidelity, and/or decrease off-target effects include N497A, R661A,Q695A, K810A, K848A, K855A, Q926A, K1003A, R1060A, and/or D1135E (withreference to the numbering system of SpyCas9).

In alternative embodiments, the Cas protein may be from a Type ICRISPR/Cas system. In some embodiments, the Cas protein may be acomponent of the Cascade complex of a Type-I CRISPR/Cas system. Forexample, the Cas protein may be a Cas3 protein. In some embodiments, theCas protein may be from a Type III CRISPR/Cas system. In someembodiments, the Cas protein may be from a Type IV CRISPR/Cas system. Insome embodiments, the Cas protein may be from a Type V CRISPR/Cassystem. In some embodiments, the Cas protein may be from a Type VICRISPR/Cas system. In some embodiments, the Cas protein may have an RNAcleavage activity. In various embodiments, the Cas protein may beclassified as Cas9, Cas12a (a.k.a. Cpf1), Cas12b, Cas12c, Cas12d, Cas12e(a.k.a. CasX), Cas13a, or Cas13b.

(i) Heterologous Domains

The Cas9 protein can be engineered to comprise at least one heterologousdomain, i.e., Cas9 is fused to one or more heterologous domains. Insituations in which two or more heterologous domains are fused withCas9, the two or more heterologous domains can be the same or they canbe different. The one or more heterologous domains can be fused to the Nterminal end, the C terminal end, an internal location, or combinationthereof. The fusion can be direct via a chemical bond, or the linkagecan be indirect via one or more linkers.

In certain preferred embodiments, the engineered Cas9 proteins describedherein include one or more nuclear localization signals (NLS).Non-limiting examples of nuclear localization signals include PKKKRKV(SEQ ID NO:1), PKKKRRV (SEQ ID NO:2), KRPAATKKAGQAKKKK (SEQ ID NO:3),YGRKKRRQRRR (SEQ ID NO:4), RKKRRQRRR (SEQ ID NO:5), PAAKRVKLD (SEQ IDNO:6), RQRRNELKRSP (SEQ ID NO:7), VSRKRPRP (SEQ ID NO:8), PPKKARED (SEQID NO:9), PQPKKKPL (SEQ ID NO:10), SALIKKKKKMAP (SEQ ID NO:11), PKQKKRK(SEQ ID NO:12), RKLKKKIKKL (SEQ ID NO:13), REKKKFLKRR (SEQ ID NO:14),KRKGDEVDGVDEVAKKKSKK (SEQ ID NO:15), RKCLQAGMNLEARKTKK (SEQ ID NO:16),NQSSNFGPMKGGNFGGRSSGPYGGGGQYFAKPRNQGGY (SEQ ID NO:17), andRMRIZFKNKGKDTAELRRRRVEVSVELRKAKKDEQILKRRNV (SEQ ID NO:18).

In one particular embodiment, the nuclear localization signal isselected from PKKKRKV (SEQ ID NO:1) and PAAKRVKLD (SEQ ID NO:6). Inanother particular embodiment, the engineered Cas9 protein includes bothof PKKKRKV (SEQ ID NO:1) and PAAKRVKLD (SEQ ID NO:6). In anotherparticular embodiment, the engineered Cas9 protein includes at least twoof PKKKRKV (SEQ ID NO:1) and at least one of PAAKRVKLD (SEQ ID NO:6). Inanother particular embodiment, the engineered Cas9 protein includes twoof PKKKRKV (SEQ ID NO:1) and one of PAAKRVKLD (SEQ ID NO:6).

In these and other preferred embodiments, the engineered Cas9 proteinsinclude one or more marker domains. Marker domains include fluorescentproteins and purification or epitope tags. Suitable fluorescent proteinsinclude, without limit, green fluorescent proteins (e.g., GFP, eGFP,GFP-2, tagGFP, turboGFP, Emerald, Azami Green, Monomeric Azami Green,CopGFP, AceGFP, ZsGreen1), yellow fluorescent proteins (e.g., YFP, EYFP,Citrine, Venus, YPet, PhiYFP, ZsYellow1), blue fluorescent proteins(e.g., BFP, EBFP, EBFP2, Azurite, mKalama1, GFPuv, Sapphire,T-sapphire), cyan fluorescent proteins (e.g., ECFP, Cerulean, CyPet,AmCyan1, Midoriishi-Cyan), red fluorescent proteins (e.g., mKate,mKate2, mPlum, DsRed monomer, mCherry, mRFP1, DsRed-Express, DsRed2,DsRed-Monomer, HcRed-Tandem, HcRed1, AsRed2, eqFP611, mRasberry,mStrawberry, Jred), orange fluorescent proteins (e.g., mOrange, mKO,Kusabira-Orange, Monomeric Kusabira-Orange, mTangerine, tdTomato), orcombinations thereof. The marker domain can comprise tandem repeats ofone or more fluorescent proteins (e.g., Suntag).

In one embodiment, the marker protein is selected from the following:

Marker Protein Sequence (SEQ ID NO: 19)MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSKLSKDPNEKRDHMVLLEFVTAAGITLGMDELYK (SEQ ID NO: 20)MVSKGEAVIKEFMRFKVHMEGSMNGHEFEIEGEGEGRPYEGTQTAKLKVTKGGPLPFSWDILSPQFMYGSRAFTKHPADIPDYYKQSFPEGFKWERVMNFEDGGAVTVTQDTSLEDGTLIYKVKLRGTNFPPDGPVMQKKTMGWEASTERLYPEDGVLKGDIKMALRLKDGGRYLADFKTTYKAKKPVQMPGAYNVDRKLDITSHNEDYTVVEQYERSEGRHSTGGMDELYK

Non-limiting examples of suitable purification or epitope tags include6×His (SEQ ID NO:22), FLAG® (e.g., SEQ ID NO:21), HA, GST, Myc, SAM, andthe like. Non-limiting examples of heterologous fusions which facilitatedetection or enrichment of CRISPR complexes include streptavidin(Kipriyanov et al., Human Antibodies, 1995, 6(3):93-101.), avidin(Airenne et al., Biomolecular Engineering, 1999, 16(1-4):87-92),monomeric forms of avidin (Laitinen et al., Journal of BiologicalChemistry, 2003, 278(6):4010-4014), peptide tags which facilitatebiotinylation during recombinant production (Cull et al., Methods inEnzymology, 2000, 326:430-440).

In addition to a nuclear localization signal(s) and a marker protein(s),in various embodiments the engineered Cas9 protein may also include oneor more heterologous domains such as a cell-penetrating domain, a markerdomain, a chromatin disrupting domain, an epigenetic modification domain(e.g., a cytidine deaminase domain, a histone acetyltransferase domain,and the like), a transcriptional regulation domain, an RNA aptamerbinding domain, or a non-Cas9 nuclease domain.

In some embodiments, the one or more heterologous domains can be acell-penetrating domain. Examples of suitable cell-penetrating domainsinclude, without limit, GRKKRRQRRRPPQPKKKRKV (SEQ ID NO:23),PLSSIFSRIGDPPKKKRKV (SEQ ID NO:24), GALFLGWLGAAGSTMGAPKKKRKV (SEQ IDNO:25), GALFLGFLGAAGSTMGAWSQPKKKRKV (SEQ ID NO:26),KETWWETWWVTEWSQPKKKRKV (SEQ ID NO:27), YARAAARQARA (SEQ ID NO:28),THRLPRRRRRR (SEQ ID NO:29), GGRRARRRRRR (SEQ ID NO:30), RRQRRTSKLMKR(SEQ ID NO:31), GWTLNSAGYLLGKINLKALAALAKKIL (SEQ ID NO:32),KALAWEAKLAKALAKALAKHLAKALAKALKCEA (SEQ ID NO:33), and RQIKIWFQNRRMKWKK(SEQ ID NO:34).

In still other embodiments, the one or more heterologous domain can be achromatin modulating motif (CMM). Non-limiting examples of CMMs includenucleosome interacting peptides derived from high mobility group (HMG)proteins (e.g., HMGB1, HMGB2, HMGB3, HMGN1, HMGN2, HMGN3a, HMGN3b,HMGN4, and HMGN5 proteins), the central globular domain of histone H1variants (e.g., histone H1.0, H1.1, H1.2, H1.3, H1.4, H1.5, H1.6, H1.7,H1.8, H1.9, and H.1.10), or DNA binding domains of chromatin remodelingcomplexes (e.g., SWI/SNF (SWItch/Sucrose Non-Fermentable), ISWI(Imitation SWItch), CHD (Chromodomain-Helicase-DNA binding), Mi-2/NuRD(Nucleosome Remodeling and Deacetylase), INO80, SWR1, and RSC complexes.In other embodiments, CMMs also can be derived from topoisomerases,helicases, or viral proteins. The source of the CMM can and will vary.CMMs can be from humans, animals (i.e., vertebrates and invertebrates),plants, algae, or yeast. Non-limiting examples of specific CMMs arelisted in the table below. Persons of skill in the art can readilyidentify homologs in other species and/or the relevant fusion motiftherein.

Accession Protein No. Fusion Motif Human HMGN1 P05114 Full length HumanHMGN2 P05204 Full length Human HMGN3a Q15651 Full length Human HMGN3bQ15651-2 Full length Human HMGN4 O00479 Full length Human HMGN5 P82970Nucleosome binding motif Human HMGB1 P09429 Box A Human histone H1.0P07305 Globular motif Human histone H1.2 P16403 Globular motif HumanCHD1 O14646 DNA binding motif Yeast CHD1 P32657 DNA binding motif YeastISWI P38144 DNA binding motif Human TOP1 P11387 DNA binding motif Humanherpesvirus 8 LANA J9QSF0 Nucleosome binding motif Human CMV IE1 P13202Chromatin tethering motif M. leprae DNA helicase P40832 HhH bindingmotif

In yet other embodiments, the one or more heterologous domains can be anepigenetic modification domain. Non-limiting examples of suitableepigenetic modification domains include those with DNA deamination(e.g., cytidine deaminase, adenosine deaminase, guanine deaminase), DNAmethyltransferase activity (e.g., cytosine methyltransferase), DNAdemethylase activity, DNA amination, DNA oxidation activity, DNAhelicase activity, histone acetyltransferase (HAT) activity (e.g., HATdomain derived from E1A binding protein p300), histone deacetylaseactivity, histone methyltransferase activity, histone demethylaseactivity, histone kinase activity, histone phosphatase activity, histoneubiquitin ligase activity, histone deubiquitinating activity, histoneadenylation activity, histone deadenylation activity, histoneSUMOylating activity, histone deSUMOylating activity, histoneribosylation activity, histone deribosylation activity, histonemyristoylation activity, histone demyristoylation activity, histonecitrullination activity, histone alkylation activity, histonedealkylation activity, or histone oxidation activity. In specificembodiments, the epigenetic modification domain can comprise cytidinedeaminase activity, adenosine deaminase activity, histoneacetyltransferase activity, or DNA methyltransferase activity.

In other embodiments, the one or more heterologous domains can be atranscriptional regulation domain (i.e., a transcriptional activationdomain or transcriptional repressor domain). Suitable transcriptionalactivation domains include, without limit, herpes simplex virus VP16domain, VP64 (i.e., four tandem copies of VP16), VP160 (i.e., ten tandemcopies of VP16), NFκB p65 activation domain (p65), Epstein-Barr virus Rtransactivator (Rta) domain, VPR (i.e., VP64+p65+Rta), p300-dependenttranscriptional activation domains, p53 activation domains 1 and 2,heat-shock factor 1 (HSF1) activation domains, Smad4 activation domains(SAD), cAMP response element binding protein (CREB) activation domains,E2A activation domains, nuclear factor of activated T-cells (NFAT)activation domains, or combinations thereof. Non-limiting examples ofsuitable transcriptional repressor domains include Kruppel-associatedbox (KRAB) repressor domains, Mxi repressor domains, inducible cAMPearly repressor (ICER) domains, YY1 glycine rich repressor domains,Sp1-like repressors, E(spI) repressors, IκB repressors, Sin3 repressors,methyl-CpG binding protein 2 (MeCP2) repressors, or combinationsthereof. Transcriptional activation or transcriptional repressor domainscan be genetically fused to the Cas9 protein or bound via noncovalentprotein-protein, protein-RNA, or protein-DNA interactions.

In further embodiments, the one or more heterologous domains can be anRNA aptamer binding domain (Konermann et al., Nature, 2015,517(7536):583-588; Zalatan et al., Cell, 2015, 160(1-2):339-50).Examples of suitable RNA aptamer protein domains include MS2 coatprotein (MCP), PP7 bacteriophage coat protein (PCP), Mu bacteriophageCom protein, lambda bacteriophage N22 protein, stem-loop binding protein(SLBP), Fragile X mental retardation syndrome-related protein 1 (FXR1),proteins derived from bacteriophage such as AP205, BZ13, f1, f2, fd, fr,ID2, JP34/GA, JP501, JP34, JP500, KU1, M11, M12, MX1, NL95, PP7, ϕCb5,ϕCb8r, ϕCb12r, ϕCb23r, Qβ, R17, SP-β, TW18, TW19, and VK, fragmentsthereof, or derivatives thereof.

In yet other embodiments, the one or more heterologous domains can be anon-Cas9 nuclease domain. Suitable nuclease domains can be obtained fromany endonuclease or exonuclease. Non-limiting examples of endonucleasesfrom which a nuclease domain can be derived include, but are not limitedto, restriction endonucleases and homing endonucleases. In someembodiments, the nuclease domain can be derived from a type II-Srestriction endonuclease. Type II-S endonucleases cleave DNA at sitesthat are typically several base pairs away from the recognition/bindingsite and, as such, have separable binding and cleavage domains. Theseenzymes generally are monomers that transiently associate to form dimersto cleave each strand of DNA at staggered locations. Non-limitingexamples of suitable type II-S endonucleases include BfiI, BpmI, BsaI,BsgI, BsmBI, BsmI, BspMI, FokI, MboII, and SapI. In some embodiments,the nuclease domain can be a FokI nuclease domain or a derivativethereof. The type II-S nuclease domain can be modified to facilitatedimerization of two different nuclease domains. For example, thecleavage domain of FokI can be modified by mutating certain amino acidresidues. By way of non-limiting example, amino acid residues atpositions 446, 447, 479, 483, 484, 486, 487, 490, 491, 496, 498, 499,500, 531, 534, 537, and 538 of FokI nuclease domains are targets formodification. In specific embodiments, the FokI nuclease domain cancomprise a first FokI half-domain comprising Q486E, I499L, and/or N496Dmutations, and a second FokI half-domain comprising E490K, I538K, and/orH537R mutations.

The one or more heterologous domains can be linked directly to the Cas9protein via one or more chemical bonds (e.g., covalent bonds), or theone or more heterologous domains can be linked indirectly to the Cas9protein via one or more linkers.

A linker is a chemical group that connects one or more other chemicalgroups via at least one covalent bond. Suitable linkers include aminoacids, peptides, nucleotides, nucleic acids, organic linker molecules(e.g., maleimide derivatives, N-ethoxybenzylimidazole,biphenyl-3,4′,5-tricarboxylic acid, p-aminobenzyloxycarbonyl, and thelike), disulfide linkers, and polymer linkers (e.g., PEG). The linkercan include one or more spacing groups including, but not limited toalkylene, alkenylene, alkynylene, alkyl, alkenyl, alkynyl, alkoxy, aryl,heteroaryl, aralkyl, aralkenyl, aralkynyl and the like. The linker canbe neutral, or carry a positive or negative charge. Additionally, thelinker can be cleavable such that the linker's covalent bond thatconnects the linker to another chemical group can be broken or cleavedunder certain conditions, including pH, temperature, salt concentration,light, a catalyst, or an enzyme. In some embodiments, the linker can bea peptide linker. The peptide linker can be a flexible amino acid linker(e.g., comprising small, non-polar or polar amino acids).

In one particular embodiment, the linker is selected from the following:

Linker Protein Sequence (SEQ ID NO: 35)AEAAAKEAAAKEAAAKEAAAKALEAEAAAKEAAAKEAAAKEAAAKA (SEQ ID NO: 36)SGGSSGGSSGSETPGTSESATPESSGGSSGGS

Other non-limiting examples of flexible linkers include LEGGGS (SEQ IDNO:37), TGSG (SEQ ID NO:38), GGSGGGSG (SEQ ID NO:39), (GGGGS)₁₋₄ (SEQ IDNO:40), and (Gly)₆₋₈ (SEQ ID NO:41). Alternatively, the peptide linkercan be a rigid amino acid linker. Such linkers include (EAAAK)₁₋₄ (SEQID NO:42), A(EAAAK)₂₋₅A (SEQ ID NO:43), PAPAP (SEQ ID NO:44), and(AP)₆₋₈ (SEQ ID NO:45). Additional examples of suitable linkers are wellknown in the art and programs to design linkers are readily available(Crasto et al., Protein Eng., 2000, 13(5):309-312).

In some embodiments, the engineered Cas9 proteins can be producedrecombinantly in cell-free systems, bacterial cells, or eukaryotic cellsand purified using standard purification means. In other embodiments,the engineered Cas9 proteins are produced in vivo in eukaryotic cells ofinterest from nucleic acids encoding the engineered Cas9 proteins (seesection (II) below).

In embodiments in which the engineered Cas9 protein comprises nucleaseor nickase activity, the engineered Cas9 protein can further comprise atleast cell-penetrating domain, as well as at least one chromatindisrupting domain. In embodiments in which the engineered Cas9 proteinis linked to an epigenetic modification domain, the engineered Cas9protein can further comprise at least one cell-penetrating domain, aswell as at least one chromatin disrupting domain. Furthermore, inembodiments in which the engineered Cas9 protein is linked to atranscriptional regulation domain, the engineered Cas9 protein canfurther comprise at least one cell-penetrating domain, as well as atleast one chromatin disrupting domain and/or at least one RNA aptamerbinding domain.

The various fusion protein components can be combined, from N-terminusto C-terminus, in any order. For example, wherein A represents themarker protein, B represents a nuclear localization signal, and Crepresents the Cas9 protein, the fusion protein can be arranged, fromN-terminus to C-terminus, in the following manner: A-B-C; A-C-B; B-A-C;B-C-A; C-A-B; or C-B-A, wherein a linker (“-L-”) may be disposed betweenany two items (e.g., A-L-B-C; A-B-L-C; A-L-B-L-C; and so on).

(b) Engineered Guide RNAs

The engineered guide RNAs is designed to complex with a specificengineered Cas9 protein. A guide RNA comprises (i) a CRISPR RNA (crRNA)that contains a guide sequence at the 5′ end that hybridizes with atarget sequence and (ii) a transacting crRNA (tracrRNA) sequence thatrecruits the Cas9 protein. The crRNA guide sequence of each guide RNA isdifferent (i.e., is sequence specific). The tracrRNA sequence isgenerally the same in guide RNAs designed to complex with a Cas9 proteinfrom a particular bacterial species.

The crRNA guide sequence is designed to hybridize with a target sequence(i.e., protospacer) in a double-stranded sequence. In general, thecomplementarity between the crRNA and the target sequence is at least80%, at least 85%, at least 90%, at least 95%, or at least 99%. Inspecific embodiments, the complementarity is complete (i.e., 100%). Invarious embodiments, the length of the crRNA guide sequence can rangefrom about 15 nucleotides to about 25 nucleotides. For example, thecrRNA guide sequence can be about 15, 16, 17, 18, 19, 20, 21, 22, 23,24, or 25 nucleotides in length. In specific embodiments, the crRNA isabout 19, 20, or 21 nucleotides in length. In one embodiment, the crRNAguide sequence has a length of 20 nucleotides.

The guide RNA comprises repeat sequence that forms at least one stemloop structure, which interacts with the Cas9 protein, and 3′ sequencethat remains single-stranded. The length of each loop and stem can vary.For example, the loop can range from about 3 to about 10 nucleotides inlength, and the stem can range from about 6 to about 20 base pairs inlength. The stem can comprise one or more bulges of 1 to about 10nucleotides. The length of the single-stranded 3′ region can vary. ThetracrRNA sequence in the engineered guide RNA generally is based uponthe coding sequence of wild type tracrRNA in the bacterial species ofinterest. The wild-type sequence can be modified to facilitate secondarystructure formation, increased secondary structure stability, facilitateexpression in eukaryotic cells, and so forth. For example, one or morenucleotide changes can be introduced into the guide RNA coding sequence(see Example 3, below). The tracrRNA sequence can range in length fromabout 50 nucleotides to about 300 nucleotides. In various embodiments,the tracrRNA can range in length from about 50 to about 90 nucleotides,from about 90 to about 110 nucleotides, from about 110 to about 130nucleotides, from about 130 to about 150 nucleotides, from about 150 toabout 170 nucleotides, from about 170 to about 200 nucleotides, fromabout 200 to about 250 nucleotides, or from about 250 to about 300nucleotides.

In general, the engineered guide RNA is a single molecule (i.e., asingle guide RNA or sgRNA), wherein the crRNA sequence is linked to thetracrRNA sequence. In some embodiments, however, the engineered guideRNA can be two separate molecules. A first molecule comprising the crRNAthat contains 3′ sequence (comprising from about 6 to about 20nucleotides) that is capable of base pairing with the 5′ end of a secondmolecule, wherein the second molecule comprises the tracrRNA thatcontains 5′ sequence (comprising from about 6 to about 20 nucleotides)that is capable of base pairing with the 3′ end of the first molecule.

In some embodiments, the tracrRNA sequence of the engineered guide RNAcan be modified to comprise one or more aptamer sequences (Konermann etal., Nature, 2015, 517(7536):583-588; Zalatan et al., Cell, 2015,160(1-2):339-50). Suitable aptamer sequences include those that bindadaptor proteins chosen from MCP, PCP, Com, SLBP, FXR1, AP205, BZ13, f1,f2, fd, fr, ID2, JP34/GA, JP501, JP34, JP500, KU1, M11, M12, MX1, NL95,PP7, ϕCb5, ϕCb8r, ϕCb12r, ϕCb23r, Qβ, R17, SP-β, TW18, TW19, VK,fragments thereof, or derivatives thereof. Those of skill in the artappreciate that the length of the aptamer sequence can vary.

In other embodiments, the guide RNA can further comprise at least onedetectable label. The detectable label can be a fluorophore (e.g., FAM,TMR, Cy3, Cy5, Texas Red, Oregon Green, Alexa Fluors, Halo tags, orsuitable fluorescent dye), a detection tag (e.g., biotin, digoxigenin,and the like), quantum dots, or gold particles.

The guide RNA can comprise standard ribonucleotides and/or modifiedribonucleotides. In some embodiment, the guide RNA can comprise standardor modified deoxyribonucleotides. In embodiments in which the guide RNAis enzymatically synthesized (i.e., in vivo or in vitro), the guide RNAgenerally comprises standard ribonucleotides. In embodiments in whichthe guide RNA is chemically synthesized, the guide RNA can comprisestandard or modified ribonucleotides and/or deoxyribonucleotides.Modified ribonucleotides and/or deoxyribonucleotides include basemodifications (e.g., pseudouridine, 2-thiouridine, N6-methyladenosine,and the like) and/or sugar modifications (e.g., 2′-O-methy, 2′-fluoro,2′-amino, locked nucleic acid (LNA), and so forth). The backbone of theguide RNA can also be modified to comprise phosphorothioate linkages,boranophosphate linkages, or peptide nucleic acids.

(c) PAM Sequence

In some embodiments, the target sequence may be adjacent to aprotospacer adjacent motif (PAM), a short sequence recognized by aCRISPR/Cas9 complex. In some embodiments, the PAM may be adjacent to orwithin 1, 2, 3, or 4, nucleotides of the 3′ end of the target sequence.The length and the sequence of the PAM may depend on the Cas9 proteinused. For example, the PAM may be selected from a consensus or aparticular PAM sequence for a specific Cas9 protein or Cas9 ortholog,including those disclosed in FIG. 1 of Ran et al., Nature, 520: 186-191(2015), which is incorporated herein by reference. In some embodiments,the PAM may comprise 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides inlength. Non-limiting exemplary PAM sequences include NGG, NGGNG, NG,NAAAAN, NNAAAAW, NNNNACA, GNNNCNNA, and NNNNGATT (wherein N is definedas any nucleotide, and W is defined as either A or T). In someembodiments, the PAM sequence may be NGG. In some embodiments, the PAMsequence may be NGGNG. In some embodiments, the PAM sequence may beNNAAAAW.

It will be understood that different CRISPR proteins recognize differentPAM sequences. For example, PAM sequences for Cas9 proteins include5′-NGG, 5′-NGGNG, 5′-NNAGAAW, 5′-NNNNGATT, 5-NNNNRYAC, 5′-NNNNCAAA,5′-NGAAA, 5′-NNAAT, 5′-NNNRTA, 5′-NNGG, 5′-NNNRTA, 5′-MMACCA,5′-NNNNGRY, 5′-NRGNK, 5′-GGGRG, 5′-NNAMMMC, and 5′-NNG, and PAMsequences for Cas12a proteins include 5′-TTN and 5′-TTTV, wherein N isdefined as any nucleotide, R is defined as either G or A, W is definedas either A or T, Y is defined an either C or T, and V is defined as A,C, or G. In general, Cas9 PAMs are located 3′ of the target sequence,and Cas12a PAMs are located 5′ of the target sequence. Various PAMsequences and the CRISPR proteins that recognize them are known in theart, e.g., U.S. Patent Application Publication 2019/0249200; Leenay,Ryan T., et al. “Identifying and visualizing functional PAM diversityacross CRISPR-Cas systems.” Molecular cell 62.1 (2016): 137-147; andKleinstiver, Benjamin P., et al. “Engineered CRISPR-Cas9 nucleases withaltered PAM specificities.” Nature 523.7561 (2015): 481, each of whichare incorporated by reference herein in their entirety.

Additionally or alternatively, the PAM for each of the engineered Cas9systems disclosed herein is presented below.

PAM Sequences PAM Engineered Cas9system (5′-3′)*Bacillus smithii Cas9 (BsmCas9) NNNNCAAALactobacillus rhamnosus Cas9 (LrhCas9) NGAAAParasutterella excrementihominis Cas9 NGG (PexCas9)Mycoplasma canis Cas9 (McaCas9) NNGGMycoplasma gallisepticum Cas9 (MgaCas9) NNAATAkkermansia glycaniphila Cas9 (AglCas9) NNNRTAAkkermansia muciniphila Cas9 (AmuCas9) MMACCAOenococcus kitaharae Cas9 (OkiCas9) NNGBifidobacterium bombi Cas9 (BboCas9) NNNNGRYAcidothermus cellulolyticus Cas9 NGG (AceCas9)Alicyclobacillus hesperidum Cas9 NGG (AheCas9)Wolinella succinogenes Cas9 (WsuCas9) NGGNitratifractor salsuginis Cas9 (NsaCas9) NRGNKRalstonia syzygii Cas9 (RsyCas9) GGGRGCorynebacterium diphtheria Cas9 (CdiCas9) NNAMMMC *K is G or T; M is Aor C; R is A or G; Y is C or T; and N is A, C, G, or T.See, e.g., U.S. Patent Application Publication No. 2019/0249200 (herebyincorporated by reference herein in its entirety.

(II) Nucleic Acids

A further aspect of the present disclosure provides nucleic acidsencoding the engineered Cas9 systems described above in section (I). Thesystems can be encoded by single nucleic acids or multiple nucleicacids. The nucleic acids can be DNA or RNA, linear or circular,single-stranded or double-stranded. The RNA or DNA can be codonoptimized for efficient translation into protein in the eukaryotic cellof interest. Codon optimization programs are available as freeware orfrom commercial sources.

In some embodiments, nucleic acid encodes a protein having at leastabout 75%, at least about 80%, at least about 85%, at least about 90%,at least about 95%, or at least about 99% sequence identity to the aminoacid sequence of SEQ ID NO:48, 49, or 50. In certain embodiments, thenucleic acid encoding the engineered Cas9 protein can have at leastabout 75%, at least about 80%, at least about 85%, at least about 90%,at least about 95%, or at least about 99% sequence identity to the DNAsequence of SEQ ID NO:48, 49, or 50. In certain embodiments, the DNAencoding the engineered Cas9 protein has the DNA sequence of SEQ IDNO:48, 49, or 50. In additional embodiments, the nucleic acid encodes aprotein having at least about 75%, at least about 80%, at least about85%, at least about 90%, at least about 95%, or at least about 99%sequence identity to the amino acid sequence of SEQ ID NO:48, 49, or 50.

In some embodiments, the nucleic acid encoding the engineered Cas9protein can be RNA. The RNA can be enzymatically synthesized in vitro.For this, DNA encoding the engineered Cas9 protein can be operablylinked to a promoter sequence that is recognized by a phage RNApolymerase for in vitro RNA synthesis. For example, the promotersequence can be a T7, T3, or SP6 promoter sequence or a variation of aT7, T3, or SP6 promoter sequence. The DNA encoding the engineeredprotein can be part of a vector, as detailed below. In such embodiments,the in vitro-transcribed RNA can be purified, capped, and/orpolyadenylated. In other embodiments, the RNA encoding the engineeredCas9 protein can be part of a self-replicating RNA (Yoshioka et al.,Cell Stem Cell, 2013, 13:246-254). The self-replicating RNA can bederived from a noninfectious, self-replicating Venezuelan equineencephalitis (VEE) virus RNA replicon, which is a positive-sense,single-stranded RNA that is capable of self-replicating for a limitednumber of cell divisions, and which can be modified to code proteins ofinterest (Yoshioka et al., Cell Stem Cell, 2013, 13:246-254).

In other embodiments, the nucleic acid encoding the engineered Cas9protein can be DNA. The DNA coding sequence can be operably linked to atleast one promoter control sequence for expression in the cell ofinterest. In certain embodiments, the DNA coding sequence can beoperably linked to a promoter sequence for expression of the engineeredCas9 protein in bacterial (e.g., E. coli) cells or eukaryotic (e.g.,yeast, insect, or mammalian) cells. Suitable bacterial promotersinclude, without limit, T7 promoters, lac operon promoters, trppromoters, tac promoters (which are hybrids of trp and lac promoters),variations of any of the foregoing, and combinations of any of theforegoing. Non-limiting examples of suitable eukaryotic promotersinclude constitutive, regulated, or cell- or tissue-specific promoters.Suitable eukaryotic constitutive promoter control sequences include, butare not limited to, cytomegalovirus immediate early promoter (CMV),simian virus (SV40) promoter, adenovirus major late promoter, Roussarcoma virus (RSV) promoter, mouse mammary tumor virus (MMTV) promoter,phosphoglycerate kinase (PGK) promoter, elongation factor (ED1)-alphapromoter, ubiquitin promoters, actin promoters, tubulin promoters,immunoglobulin promoters, fragments thereof, or combinations of any ofthe foregoing. Examples of suitable eukaryotic regulated promotercontrol sequences include without limit those regulated by heat shock,metals, steroids, antibiotics, or alcohol. Non-limiting examples oftissue-specific promoters include B29 promoter, CD14 promoter, CD43promoter, CD45 promoter, CD68 promoter, desmin promoter, elastase-1promoter, endoglin promoter, fibronectin promoter, Flt-1 promoter, GFAPpromoter, GPIIb promoter, ICAM-2 promoter, INF-β promoter, Mb promoter,NphsI promoter, OG-2 promoter, SP-B promoter, SYN1 promoter, and WASPpromoter. The promoter sequence can be wild type or it can be modifiedfor more efficient or efficacious expression. In some embodiments, theDNA coding sequence also can be linked to a polyadenylation signal(e.g., SV40 polyA signal, bovine growth hormone (BGH) polyA signal,etc.) and/or at least one transcriptional termination sequence. In somesituations, the engineered Cas9 protein can be purified from thebacterial or eukaryotic cells.

In still other embodiments, the engineered guide RNA can be encoded byDNA. In some instances, the DNA encoding the engineered guide RNA can beoperably linked to a promoter sequence that is recognized by a phage RNApolymerase for in vitro RNA synthesis. For example, the promotersequence can be a T7, T3, or SP6 promoter sequence or a variation of aT7, T3, or SP6 promoter sequence. In other instances, the DNA encodingthe engineered guide RNA can be operably linked to a promoter sequencethat is recognized by RNA polymerase III (Pol III) for expression ineukaryotic cells of interest. Examples of suitable Pol III promotersinclude, but are not limited to, mammalian U6, U3, H1, and 7SL RNApromoters.

In various embodiments, the nucleic acid encoding the engineered Cas9protein can be present in a vector. In some embodiments, the vector canfurther comprise nucleic acid encoding the engineered guide RNA.Suitable vectors include plasmid vectors, viral vectors, andself-replicating RNA (Yoshioka et al., Cell Stem Cell, 2013,13:246-254). In some embodiments, the nucleic acid encoding the complexor fusion protein can be present in a plasmid vector. Non-limitingexamples of suitable plasmid vectors include pUC, pBR322, pET,pBluescript, and variants thereof. In other embodiments, the nucleicacid encoding the complex or fusion protein can be part of a viralvector (e.g., lentiviral vectors, adeno-associated viral vectors,adenoviral vectors, and so forth). The plasmid or viral vector cancomprise additional expression control sequences (e.g., enhancersequences, Kozak sequences, polyadenylation sequences, transcriptionaltermination sequences, etc.), selectable marker sequences (e.g.,antibiotic resistance genes), origins of replication, and the like.Additional information about vectors and use thereof can be found in“Current Protocols in Molecular Biology” Ausubel et al., John Wiley &Sons, New York, 2003 or “Molecular Cloning: A Laboratory Manual”Sambrook & Russell, Cold Spring Harbor Press, Cold Spring Harbor, N.Y.,3^(rd) edition, 2001.

(III) Eukaryotic Cells

Another aspect of the present disclosure comprises eukaryotic cellscomprising at least one engineered Cas9 system as detailed above insection (I) and/or at least one nucleic acid encoding and engineeredCas9 protein and/or engineered guide RNA as detailed above in section(II).

The eukaryotic cell can be a human cell, a non-human mammalian cell, anon-mammalian vertebrate cell, an invertebrate cell, a plant cell, or asingle cell eukaryotic organism. Examples of suitable eukaryotic cellsare detailed below in section (IV)(c). The eukaryotic cell can be invitro, ex vivo, or in vivo.

By way of example, in some embodiments, the eukaryotic cell, or apopulation of eukaryotic cells, is a T-cell, a CD8⁺ T-cell, a CD8⁺ naiveT cell, a central memory T cell, an effector memory T-cell, a CD4⁺T-cell, a stem cell memory T-cell, a helper T-cell, a regulatory T-cell,a cytotoxic T-cell, a natural killer T-cell, a hematopoietic stem cell,a long term hematopoietic stem cell, a short term hematopoietic stemcell, a multipotent progenitor cell, a lineage restricted progenitorcell, a lymphoid progenitor cell, a pancreatic progenitor cell, anendocrine progenitor cell, an exocrine progenitor cell, a myeloidprogenitor cell, a common myeloid progenitor cell, an erythroidprogenitor cell, a megakaryocyte erythroid progenitor cell, a monocyticprecursor cell, an endocrine precursor cell, an exocrine cell, afibroblast, a hepatoblast, a myoblast, a macrophage, an islet beta-cell,a cardiomyocyte, a blood cell, a ductal cell, an acinar cell, an alphacell, a beta cell, a delta cell, a PP cell, a cholangiocyte, a retinalcell, a photoreceptor cell, a rod cell, a cone cell, a retinal pigmentedepithelium cell, a trabecular meshwork cell, a cochlear hair cell, anouter hair cell, an inner hair cell, a pulmonary epithelial cell, abronchial epithelial cell, an alveolar epithelial cell, a pulmonaryepithelial progenitor cell, a striated muscle cell, a cardiac musclecell, a muscle satellite cell, a myocyte, a neuron, a neuronal stemcell, a mesenchymal stem cell, an induced pluripotent stem (iPS) cell,an embryonic stem cell, a monocyte, a megakaryocyte, a neutrophil, aneosinophil, a basophil, a mast cell, a reticulocyte, a B cell, e.g. aprogenitor B cell, a Pre B cell, a Pro B cell, a memory B cell, a plasmaB cell, a gastrointestinal epithelial cell, a biliary epithelial cell, apancreatic ductal epithelial cell, an intestinal stem cell, ahepatocyte, a liver stellate cell, a Kupffer cell, an osteoblast, anosteoclast, an adipocyte (e.g., a brown adipocyte, or a whiteadipocyte), a preadipocyte, a pancreatic precursor cell, a pancreaticislet cell, a pancreatic beta cell, a pancreatic alpha cell, apancreatic delta cell, a pancreatic exocrine cell, a Schwann cell, or anoligodendrocyte, or a population of such cells.

(IV) Methods for Modifying Chromosomal Sequences

A further aspect of the present disclosure encompasses methods formodifying a chromosomal sequence in eukaryotic cells. In general, themethods comprise introducing into the eukaryotic cell of interest atleast one engineered Cas9 system as detailed above in section (I) and/orat least one nucleic acid encoding said engineered Cas9 system asdetailed above in section (II).

In embodiments in which the engineered Cas9 protein comprises nucleaseor nickase activity, the chromosomal sequence modification can comprisea substitution of at least one nucleotide, a deletion of at least onenucleotide, an insertion of at least one nucleotide. In some iterations,the method comprises introducing into the eukaryotic cell one engineeredCas9 system comprising nuclease activity or two engineered Cas9 systemscomprising nickase activity and no donor polynucleotide, such that theengineered Cas9 system or systems introduce a double-stranded break inthe target site in the chromosomal sequence and repair of thedouble-stranded break by cellular DNA repair processes introduces atleast one nucleotide change (i.e., indel), thereby inactivating thechromosomal sequence (i.e., gene knock-out). In other iterations, themethod comprises introducing into the eukaryotic cell one engineeredCas9 system comprising nuclease activity or two engineered Cas9 systemscomprising nickase activity, as well as the donor polynucleotide, suchthat the engineered Cas9 system or systems introduce a double-strandedbreak in the target site in the chromosomal sequence and repair of thedouble-stranded break by cellular DNA repair processes leads toinsertion or exchange of sequence in the donor polynucleotide into thetarget site in the chromosomal sequence (i.e., gene correction or geneknock-in).

In embodiments, in which the engineered Cas9 protein comprisesepigenetic modification activity or transcriptional regulation activity,the chromosomal sequence modification can comprise a conversion of atleast one nucleotide in or near the target site, a modification of atleast one nucleotide in or near the target site, a modification of atleast one histone protein in or near the target site, and/or a change intranscription in or near the target site in the chromosomal sequence.

(a) Introduction into the Cell

As mentioned above, the method comprises introducing into the eukaryoticcell at least one engineered Cas9 system and/or nucleic acid encodingsaid system (and optional donor polynucleotide). The at least one systemand/or nucleic acid/donor polynucleotide can be introduced into the cellof interest by a variety of means.

In some embodiments, the cell can be transfected with the appropriatemolecules (i.e., protein, DNA, and/or RNA). Suitable transfectionmethods include nucleofection (or electroporation), calciumphosphate-mediated transfection, cationic polymer transfection (e.g.,DEAE-dextran or polyethylenimine), viral transduction, virosometransfection, virion transfection, liposome transfection, cationicliposome transfection, immunoliposome transfection, nonliposomal lipidtransfection, dendrimer transfection, heat shock transfection,magnetofection, lipofection, gene gun delivery, impalefection,sonoporation, optical transfection, and proprietary agent-enhanceduptake of nucleic acids. Transfection methods are well known in the art(see, e.g., “Current Protocols in Molecular Biology” Ausubel et al.,John Wiley & Sons, New York, 2003 or “Molecular Cloning: A LaboratoryManual” Sambrook & Russell, Cold Spring Harbor Press, Cold SpringHarbor, N.Y., 3rd edition, 2001). In other embodiments, the moleculescan be introduced into the cell by microinjection. For example, themolecules can be injected into the cytoplasm or nuclei of the cells ofinterest. The amount of each molecule introduced into the cell can vary,but those skilled in the art are familiar with means for determining theappropriate amount.

The various molecules can be introduced into the cell simultaneously orsequentially. For example, the engineered Cas9 system (or its encodingnucleic acid) and the donor polynucleotide can be introduced at the sametime. Alternatively, one can be introduced first and then the other canbe introduced later into the cell.

In general, the cell is maintained under conditions appropriate for cellgrowth and/or maintenance. Suitable cell culture conditions are wellknown in the art and are described, for example, in Santiago et al.,Proc. Natl. Acad. Sci. USA, 2008, 105:5809-5814; Moehle et al. Proc.Natl. Acad. Sci. USA, 2007, 104:3055-3060; Urnov et al., Nature, 2005,435:646-651; and Lombardo et al., Nat. Biotechnol., 2007, 25:1298-1306.Those of skill in the art appreciate that methods for culturing cellsare known in the art and can and will vary depending on the cell type.Routine optimization may be used, in all cases, to determine the besttechniques for a particular cell type.

(b) Optional Donor Polynucleotide

In embodiments in which the engineered Cas9 protein comprises nucleaseor nickase activity, the method can further comprise introducing atleast one donor polynucleotide into the cell. The donor polynucleotidecan be single-stranded or double-stranded, linear or circular, and/orRNA or DNA. In some embodiments, the donor polynucleotide can be avector, e.g., a plasmid vector.

The donor polynucleotide comprises at least one donor sequence. In someaspects, the donor sequence of the donor polynucleotide can be amodified version of an endogenous or native chromosomal sequence. Forexample, the donor sequence can be essentially identical to a portion ofthe chromosomal sequence at or near the sequence targeted by theengineered Cas9 system, but which comprises at least one nucleotidechange. Thus, upon integration or exchange with the native sequence, thesequence at the targeted chromosomal location comprises at least onenucleotide change. For example, the change can be an insertion of one ormore nucleotides, a deletion of one or more nucleotides, a substitutionof one or more nucleotides, or combinations thereof. As a consequence ofthe “gene correction” integration of the modified sequence, the cell canproduce a modified gene product from the targeted chromosomal sequence.

In other aspects, the donor sequence of the donor polynucleotide can bean exogenous sequence. As used herein, an “exogenous” sequence refers toa sequence that is not native to the cell, or a sequence whose nativelocation is in a different location in the genome of the cell. Forexample, the exogenous sequence can comprise protein coding sequence,which can be operably linked to an exogenous promoter control sequencesuch that, upon integration into the genome, the cell is able to expressthe protein coded by the integrated sequence. Alternatively, theexogenous sequence can be integrated into the chromosomal sequence suchthat its expression is regulated by an endogenous promoter controlsequence. In other iterations, the exogenous sequence can be atranscriptional control sequence, another expression control sequence,an RNA coding sequence, and so forth. As noted above, integration of anexogenous sequence into a chromosomal sequence is termed a “knock in.”

As can be appreciated by those skilled in the art, the length of thedonor sequence can and will vary. For example, the donor sequence canvary in length from several nucleotides to hundreds of nucleotides tohundreds of thousands of nucleotides.

Typically, the donor sequence in the donor polynucleotide is flanked byan upstream sequence and a downstream sequence, which have substantialsequence identity to sequences located upstream and downstream,respectively, of the sequence targeted by the engineered Cas9 system.Because of these sequence similarities, the upstream and downstreamsequences of the donor polynucleotide permit homologous recombinationbetween the donor polynucleotide and the targeted chromosomal sequencesuch that the donor sequence can be integrated into (or exchanged with)the chromosomal sequence.

The upstream sequence, as used herein, refers to a nucleic acid sequencethat shares substantial sequence identity with a chromosomal sequenceupstream of the sequence targeted by the engineered Cas9 system.Similarly, the downstream sequence refers to a nucleic acid sequencethat shares substantial sequence identity with a chromosomal sequencedownstream of the sequence targeted by the engineered Cas9 system. Asused herein, the phrase “substantial sequence identity” refers tosequences having at least about 75% sequence identity. Thus, theupstream and downstream sequences in the donor polynucleotide can haveabout 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%,88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequenceidentity with sequence upstream or downstream to the target sequence. Inan exemplary embodiment, the upstream and downstream sequences in thedonor polynucleotide can have about 95% or 100% sequence identity withchromosomal sequences upstream or downstream to the sequence targeted bythe engineered Cas9 system.

In some embodiments, the upstream sequence shares substantial sequenceidentity with a chromosomal sequence located immediately upstream of thesequence targeted by the engineered Cas9 system. In other embodiments,the upstream sequence shares substantial sequence identity with achromosomal sequence that is located within about one hundred (100)nucleotides upstream from the target sequence. Thus, for example, theupstream sequence can share substantial sequence identity with achromosomal sequence that is located about 1 to about 20, about 21 toabout 40, about 41 to about 60, about 61 to about 80, or about 81 toabout 100 nucleotides upstream from the target sequence. In someembodiments, the downstream sequence shares substantial sequenceidentity with a chromosomal sequence located immediately downstream ofthe sequence targeted by the engineered Cas9 system. In otherembodiments, the downstream sequence shares substantial sequenceidentity with a chromosomal sequence that is located within about onehundred (100) nucleotides downstream from the target sequence. Thus, forexample, the downstream sequence can share substantial sequence identitywith a chromosomal sequence that is located about 1 to about 20, about21 to about 40, about 41 to about 60, about 61 to about 80, or about 81to about 100 nucleotides downstream from the target sequence.

Each upstream or downstream sequence can range in length from about 20nucleotides to about 5000 nucleotides. In some embodiments, upstream anddownstream sequences can comprise about 50, 100, 200, 300, 400, 500,600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700,1800, 1900, 2000, 2100, 2200, 2300, 2400, 2500, 2600, 2800, 3000, 3200,3400, 3600, 3800, 4000, 4200, 4400, 4600, 4800, or 5000 nucleotides. Inspecific embodiments, upstream and downstream sequences can range inlength from about 50 to about 1500 nucleotides.

(c) Cell Types

A variety of eukaryotic cells are suitable for use in the methodsdisclosed herein. For example, the cell can be a human cell, a non-humanmammalian cell, a non-mammalian vertebrate cell, an invertebrate cell,an insect cell, a plant cell, a yeast cell, or a single cell eukaryoticorganism. In some embodiments, the cell can be a one cell embryo. Forexample, a non-human mammalian embryo including rat, hamster, rodent,rabbit, feline, canine, ovine, porcine, bovine, equine, and primateembryos. In still other embodiments, the cell can be a stem cell such asembryonic stem cells, ES-like stem cells, fetal stem cells, adult stemcells, and the like. In one embodiment, the stem cell is not a humanembryonic stem cell. Furthermore, the stem cells may include those madeby the techniques disclosed in WO2003/046141, which is incorporatedherein in its entirety, or Chung et al. (Cell Stem Cell, 2008,2:113-117). The cell can be in vitro (i.e., in culture), ex vivo (i.e.,within tissue isolated from an organism), or in vivo (i.e., within anorganism). In exemplary embodiments, the cell is a mammalian cell ormammalian cell line. In particular embodiments, the cell is a human cellor human cell line.

By way of example, in some embodiments, the eukaryotic cell, or apopulation of eukaryotic cells, is a T-cell, a CD8⁺ T-cell, a CD8⁺ naiveT cell, a central memory T cell, an effector memory T-cell, a CD4⁺T-cell, a stem cell memory T-cell, a helper T-cell, a regulatory T-cell,a cytotoxic T-cell, a natural killer T-cell, a hematopoietic stem cell,a long term hematopoietic stem cell, a short term hematopoietic stemcell, a multipotent progenitor cell, a lineage restricted progenitorcell, a lymphoid progenitor cell, a pancreatic progenitor cell, anendocrine progenitor cell, an exocrine progenitor cell, a myeloidprogenitor cell, a common myeloid progenitor cell, an erythroidprogenitor cell, a megakaryocyte erythroid progenitor cell, a monocyticprecursor cell, an endocrine precursor cell, an exocrine cell, afibroblast, a hepatoblast, a myoblast, a macrophage, an islet beta-cell,a cardiomyocyte, a blood cell, a ductal cell, an acinar cell, an alphacell, a beta cell, a delta cell, a PP cell, a cholangiocyte, a retinalcell, a photoreceptor cell, a rod cell, a cone cell, a retinal pigmentedepithelium cell, a trabecular meshwork cell, a cochlear hair cell, anouter hair cell, an inner hair cell, a pulmonary epithelial cell, abronchial epithelial cell, an alveolar epithelial cell, a pulmonaryepithelial progenitor cell, a striated muscle cell, a cardiac musclecell, a muscle satellite cell, a myocyte, a neuron, a neuronal stemcell, a mesenchymal stem cell, an induced pluripotent stem (iPS) cell,an embryonic stem cell, a monocyte, a megakaryocyte, a neutrophil, aneosinophil, a basophil, a mast cell, a reticulocyte, a B cell, e.g. aprogenitor B cell, a Pre B cell, a Pro B cell, a memory B cell, a plasmaB cell, a gastrointestinal epithelial cell, a biliary epithelial cell, apancreatic ductal epithelial cell, an intestinal stem cell, ahepatocyte, a liver stellate cell, a Kupffer cell, an osteoblast, anosteoclast, an adipocyte (e.g., a brown adipocyte, or a whiteadipocyte), a preadipocyte, a pancreatic precursor cell, a pancreaticislet cell, a pancreatic beta cell, a pancreatic alpha cell, apancreatic delta cell, a pancreatic exocrine cell, a Schwann cell, or anoligodendrocyte, or a population of such cells.

Non-limiting examples of suitable mammalian cells or cell lines includehuman embryonic kidney cells (HEK293, HEK293T); human cervical carcinomacells (HELA); human lung cells (W138); human liver cells (Hep G2); humanU2-OS osteosarcoma cells, human A549 cells, human A-431 cells, and humanK562 cells; Chinese hamster ovary (CHO) cells, baby hamster kidney (BHK)cells; mouse myeloma NS0 cells, mouse embryonic fibroblast 3T3 cells(NIH3T3), mouse B lymphoma A20 cells; mouse melanoma B16 cells; mousemyoblast C2C12 cells; mouse myeloma SP2/0 cells; mouse embryonicmesenchymal C3H-10T1/2 cells; mouse carcinoma CT26 cells, mouse prostateDuCuP cells; mouse breast EMT6 cells; mouse hepatoma Hepa1c1c7 cells;mouse myeloma J5582 cells; mouse epithelial MTD-1A cells; mousemyocardial MyEnd cells; mouse renal RenCa cells; mouse pancreatic RIN-5Fcells; mouse melanoma X64 cells; mouse lymphoma YAC-1 cells; ratglioblastoma 9L cells; rat B lymphoma RBL cells; rat neuroblastoma B35cells; rat hepatoma cells (HTC); buffalo rat liver BRL 3A cells; caninekidney cells (MDCK); canine mammary (CMT) cells; rat osteosarcoma D17cells; rat monocyte/macrophage DH82 cells; monkey kidney SV-40transformed fibroblast (COS7) cells; monkey kidney CVI-76 cells; Africangreen monkey kidney (VERO-76) cells. An extensive list of mammalian celllines may be found in the American Type Culture Collection catalog(ATCC, Manassas, VA).

(V) Applications

The compositions and methods disclosed herein can be used in a varietyof therapeutic, diagnostic, industrial, and research applications. Insome embodiments, the present disclosure can be used to modify anychromosomal sequence of interest in a cell, animal, or plant in order tomodel and/or study the function of genes, study genetic or epigeneticconditions of interest, or study biochemical pathways involved invarious diseases or disorders. For example, transgenic organisms can becreated that model diseases or disorders, wherein the expression of oneor more nucleic acid sequences associated with a disease or disorder isaltered. The disease model can be used to study the effects of mutationson the organism, study the development and/or progression of thedisease, study the effect of a pharmaceutically active compound on thedisease, and/or assess the efficacy of a potential gene therapystrategy.

In other embodiments, the compositions and methods can be used toperform efficient and cost effective functional genomic screens, whichcan be used to study the function of genes involved in a particularbiological process and how any alteration in gene expression can affectthe biological process, or to perform saturating or deep scanningmutagenesis of genomic loci in conjunction with a cellular phenotype.Saturating or deep scanning mutagenesis can be used to determinecritical minimal features and discrete vulnerabilities of functionalelements required for gene expression, drug resistance, and reversal ofdisease, for example.

In further embodiments, the compositions and methods disclosed hereincan be used for diagnostic tests to establish the presence of a diseaseor disorder and/or for use in determining treatment options. Examples ofsuitable diagnostic tests include detection of specific mutations incancer cells (e.g., specific mutation in EGFR, HER2, and the like),detection of specific mutations associated with particular diseases(e.g., trinucleotide repeats, mutations in β-globin associated withsickle cell disease, specific SNPs, etc.), detection of hepatitis,detection of viruses (e.g., Zika), and so forth.

In additional embodiments, the compositions and methods disclosed hereincan be used to correct genetic mutations associated with a particulardisease or disorder such as, e.g., correct globin gene mutationsassociated with sickle cell disease or thalassemia, correct mutations inthe adenosine deaminase gene associated with severe combined immunedeficiency (SCID), reduce the expression of HTT, the disease-causinggene of Huntington's disease, or correct mutations in the rhodopsin genefor the treatment of retinitis pigmentosa. Such modifications may bemade in cells ex vivo.

In still other embodiments, the compositions and methods disclosedherein can be used to generate crop plants with improved traits orincreased resistance to environmental stresses. The present disclosurecan also be used to generate farm animal with improved traits orproduction animals. For example, pigs have many features that make themattractive as biomedical models, especially in regenerative medicine orxenotransplantation.

In still other embodiments, the compositions and methods disclosedherein can be used to determine chromosome identity and location withina living cell or chemically fixed cell (such as formalin fixation usedin formalin-fixed paraffin embedded clinical samples). For example, aCRIPSR complex linked via a peptide sequence disclosed herein to afluorescent protein may be targeted in single or multiple copies to agenetic locus, and such complexes detected by microscopy to determinechromosomal locus copy number and/or location. Example genetic loci fortracking might include centromeric regions, telomeric regions, or otherrepetitive regions of the genome to which multiple copies of a singleidentical CRISPR complex may bind.

Definitions

Unless defined otherwise, all technical and scientific terms used hereinhave the meaning commonly understood by a person skilled in the art towhich this invention belongs. The following references provide one ofskill with a general definition of many of the terms used in thisinvention: Singleton et al., Dictionary of Microbiology and MolecularBiology (2nd Ed. 1994); The Cambridge Dictionary of Science andTechnology (Walker ed., 1988); The Glossary of Genetics, 5th Ed., R.Rieger et al. (eds.), Springer Verlag (1991); and Hale & Marham, TheHarper Collins Dictionary of Biology (1991). As used herein, thefollowing terms have the meanings ascribed to them unless specifiedotherwise.

When introducing elements of the present disclosure or the preferredembodiments(s) thereof, the articles “a”, “an”, “the” and “said” areintended to mean that there are one or more of the elements. The terms“comprising”, “including” and “having” are intended to be inclusive andmean that there may be additional elements other than the listedelements.

The term “about” when used in relation to a numerical value, x, forexample means x±5%.

As used herein, the terms “complementary” or “complementarity” refer tothe association of double-stranded nucleic acids by base pairing throughspecific hydrogen bonds. The base paring may be standard Watson-Crickbase pairing (e.g., 5′-A G T C-3′ pairs with the complementary sequence3′-T C A G-5′). The base pairing also may be Hoogsteen or reversedHoogsteen hydrogen bonding. Complementarity is typically measured withrespect to a duplex region and thus, excludes overhangs, for example.Complementarity between two strands of the duplex region may be partialand expressed as a percentage (e.g., 70%), if only some (e.g., 70%) ofthe bases are complementary. The bases that are not complementary are“mismatched.” Complementarity may also be complete (i.e., 100%), if allthe bases in the duplex region are complementary.

As used herein, the term “CRISPR/Cas system” or “Cas9 system” refers toa complex comprising a Cas9 protein (i.e., nuclease, nickase, orcatalytically dead protein) and a guide RNA.

The term “endogenous sequence,” as used herein, refers to a chromosomalsequence that is native to the cell.

As used herein, the term “exogenous” refers to a sequence that is notnative to the cell, or a chromosomal sequence whose native location inthe genome of the cell is in a different chromosomal location.

A “gene,” as used herein, refers to a DNA region (including exons andintrons) encoding a gene product, as well as all DNA regions whichregulate the production of the gene product, whether or not suchregulatory sequences are adjacent to coding and/or transcribedsequences. Accordingly, a gene includes, but is not necessarily limitedto, promoter sequences, terminators, translational regulatory sequencessuch as ribosome binding sites and internal ribosome entry sites,enhancers, silencers, insulators, boundary elements, replicationorigins, matrix attachment sites, and locus control regions.

The term “heterologous” refers to an entity that is not endogenous ornative to the cell of interest. For example, a heterologous proteinrefers to a protein that is derived from or was originally derived froman exogenous source, such as an exogenously introduced nucleic acidsequence. In some instances, the heterologous protein is not normallyproduced by the cell of interest.

The term “nickase” refers to an enzyme that cleaves one strand of adouble-stranded nucleic acid sequence (i.e., nicks a double-strandedsequence). For example, a nuclease with double strand cleavage activitycan be modified by mutation and/or deletion to function as a nickase andcleave only one strand of a double-stranded sequence.

The term “nuclease,” as used herein, refers to an enzyme that cleavesboth strands of a double-stranded nucleic acid sequence.

The terms “nucleic acid” and “polynucleotide” refer to adeoxyribonucleotide or ribonucleotide polymer, in linear or circularconformation, and in either single- or double-stranded form. For thepurposes of the present disclosure, these terms are not to be construedas limiting with respect to the length of a polymer. The terms canencompass known analogs of natural nucleotides, as well as nucleotidesthat are modified in the base, sugar and/or phosphate moieties (e.g.,phosphorothioate backbones). In general, an analog of a particularnucleotide has the same base-pairing specificity; i.e., an analog of Awill base-pair with T.

The term “nucleotide” refers to deoxyribonucleotides or ribonucleotides.The nucleotides may be standard nucleotides (i.e., adenosine, guanosine,cytidine, thymidine, and uridine), nucleotide isomers, or nucleotideanalogs. A nucleotide analog refers to a nucleotide having a modifiedpurine or pyrimidine base or a modified ribose moiety. A nucleotideanalog may be a naturally occurring nucleotide (e.g., inosine,pseudouridine, etc.) or a non-naturally occurring nucleotide.Non-limiting examples of modifications on the sugar or base moieties ofa nucleotide include the addition (or removal) of acetyl groups, aminogroups, carboxyl groups, carboxymethyl groups, hydroxyl groups, methylgroups, phosphoryl groups, and thiol groups, as well as the substitutionof the carbon and nitrogen atoms of the bases with other atoms (e.g.,7-deaza purines). Nucleotide analogs also include dideoxy nucleotides,2′-O-methyl nucleotides, locked nucleic acids (LNA), peptide nucleicacids (PNA), and morpholinos.

The terms “polypeptide” and “protein” are used interchangeably to referto a polymer of amino acid residues.

The terms “target sequence,” “target chromosomal sequence,” and “targetsite” are used interchangeably to refer to the specific sequence inchromosomal DNA to which the engineered Cas9 system is targeted, and thesite at which the engineered Cas9 system modifies the DNA or protein(s)associated with the DNA.

Techniques for determining nucleic acid and amino acid sequence identityare known in the art. Typically, such techniques include determining thenucleotide sequence of the mRNA for a gene and/or determining the aminoacid sequence encoded thereby, and comparing these sequences to a secondnucleotide or amino acid sequence. Genomic sequences can also bedetermined and compared in this fashion. In general, identity refers toan exact nucleotide-to-nucleotide or amino acid-to-amino acidcorrespondence of two polynucleotides or polypeptide sequences,respectively. Two or more sequences (polynucleotide or amino acid) canbe compared by determining their percent identity. The percent identityof two sequences, whether nucleic acid or amino acid sequences, is thenumber of exact matches between two aligned sequences divided by thelength of the shorter sequences and multiplied by 100. An approximatealignment for nucleic acid sequences is provided by the local homologyalgorithm of Smith and Waterman, Advances in Applied Mathematics2:482-489 (1981). This algorithm can be applied to amino acid sequencesby using the scoring matrix developed by Dayhoff, Atlas of ProteinSequences and Structure, M. O. Dayhoff ed., 5 suppl. 3:353-358, NationalBiomedical Research Foundation, Washington, D.C., USA, and normalized byGribskov, Nucl. Acids Res. 14(6):6745-6763 (1986). An exemplaryimplementation of this algorithm to determine percent identity of asequence is provided by the Genetics Computer Group (Madison, Wis.) inthe “BestFit” utility application. Other suitable programs forcalculating the percent identity or similarity between sequences aregenerally known in the art, for example, another alignment program isBLAST, used with default parameters. For example, BLASTN and BLASTP canbe used using the following default parameters: genetic code=standard;filter=none; strand=both; cutoff=60; expect=10; Matrix=BLOSUM62;Descriptions=50 sequences; sort by=HIGH SCORE; Databases=non-redundant,GenBank+EMBL+DDBJ+PDB+GenBank CDS translations+Swissprotein+Spupdate+PIR. Details of these programs can be found on theGenBank website.

As various changes could be made in the above-described cells andmethods without departing from the scope of the invention, it isintended that all matter contained in the above description and in theexamples given below, shall be interpreted as illustrative and not in alimiting sense.

Examples

The following examples illustrate certain aspects of the disclosure.

Example 1: Human Cell Gene Editing Using GFP-SpCas9 and RFP-SpCas9Fusion Proteins

Human K562 cells (0.35×10⁶) were transfected with 60 pmol of SpCas9,GFP-SpCas9, or RFP-SpCas9 recombinant protein and 180 pmol of an invitro transcribed single guide RNA (sgRNA) targeting the human EMX1locus with the guide sequence 5′-GCUCCCAUCACAUCAACCGG-3′ (SEQ ID NO:65). Transfection was carried out using Nucleofection Solution V and anAmaxa instrument. Cells were maintained at 37° C. and 5% CO₂ for threedays before harvested for gene editing analysis. Genomic DNA wasprepared using QuickExtract DNA extraction solution. Targeted EMX1region was PCR amplified using primers consisting of target-specificsequences and next generation sequencing (NGS) adaptors. The forwardprimer is 5′-TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGNNNNNNAGTCTTCCCATCAGGCTCTCA-3′ (SEQ ID NO:46) and the reverse primer isGTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGNNNNNNAGAGTCCAGCTTGGG CC-3′ (SEQ IDNO:47), where the target-specific sequences are underlined, and Nrepresents A, T, G, or C. PCR amplicons were analyzed by NGS using theIllumina MiSeq to determine the editing efficiency of each Cas9 protein.The results displayed in FIG. 1 show that the GFP-SpCas9 and RFP-SpCas9fusing proteins each retain the editing activity parallel to the levelby SpCas9 protein.

Table 1 presents the human codon optimized DNA and protein sequences ofengineered Cas9/NLS proteins, wherein the NLS sequences are presented inbold text and the linker between the marker protein and Cas9 ispresented in underlined text.

TABLE 1 Engineered Cas9 SystemsAmino acid sequence of GFP-SpCas9 fusion proteinMVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSKLSKDPNEKRDHMVLLEFVTAAGITLGMDELYKVDAEAAAKEAAAKEAAAKEAAAKALEAEAAAKEAAAKEAAAKEAAA KAPAAKRVKLDGGGGSTGMDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGDEFPKKKRKVGGGGSPKKKRKV (SEQ ID NO: 48) Underlined: Linker between GFP and SpCas9Bold: Nuclear localization signalsAmino acid sequence of RFP-SpCas9 fusion proteinMVSKGEAVIKEFMRFKVHMEGSMNGHEFEIEGEGEGRPYEGTQTAKLKVTKGGPLPFSWDILSPQFMYGSRAFTKHPADIPDYYKQSFPEGFKWERVMNFEDGGAVTVTQDTSLEDGTLIYKVKLRGTNFPPDGPVMQKKTMGWEASTERLYPEDGVLKGDIKMALRLKDGGRYLADFKTTYKAKKPVQMPGAYNVDRKLDITSHNEDYTVVEQYERSEGRHSTGGMDELYKVDSGGSSGGSSGSETPGTSESATPESSGGSSGGS PAAKRVKLDGGGGSTGMDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGDEFPKKKRKVGGGGSPKKKRKV (SEQ ID NO: 49)Underlined: Linker between RFP and SpCas9Bold: Nuclear localization signalsAmino acid sequence of GFP-eSpCas9 fusion proteinMVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSKLSKDPNEKRDHMVLLEFVTAAGITLGMDELYKVDSGGSSGGSSGSETPGTSESATPESSGGSSGGS PAAKRVKLDGGGGSTGMDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLADDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPALESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKAPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGDEFPKKKRKVGGGGSPKKKRKV (SEQ ID NO: 50)Underlined: Linker between GFP and eSpCas9Bold: Nuclear localization signals

Human codon optimized DNA sequences used to produce the three proteinsare as follows:

Human codon optimized GFP-SpCas9 DNA sequence (SEQ ID NO: 62)ATGGTTAGCAAAGGTGAAGAACTGTTTACAGGTGTTGTTCCGATTCTGGTTGAACTGGATGGTGATGTTAATGGCCACAAATTTTCAGTTAGCGGTGAAGGCGAAGGTGATGCAACCTATGGTAAACTGACCCTGAAATTTATCTGTACCACCGGCAAACTGCCGGTTCCGTGGCCGACACTGGTTACCACACTGACCTATGGTGTTCAGTGTTTTAGCCGTTATCCGGATCACATGAAACAGCACGATTTTTTCAAAAGCGCAATGCCGGAAGGTTATGTTCAAGAACGTACCATCTTCTTCAAAGATGACGGCAACTATAAAACCCGTGCCGAAGTTAAATTTGAAGGTGATACCCTGGTGAATCGCATTGAACTGAAAGGCATCGATTTTAAAGAGGATGGTAATATCCTGGGCCACAAACTGGAATATAATTATAATAGCCACAACGTGTACATCATGGCCGACAAACAGAAAAATGGCATCAAAGTGAACTTCAAGATCCGCCATAATATTGAAGATGGTTCAGTTCAGCTGGCCGATCATTATCAGCAGAATACCCCGATTGGTGATGGTCCGGTTCTGCTGCCGGATAATCATTATCTGAGCACCCAGAGCAAACTGAGCAAAGATCCGAATGAAAAACGTGATCACATGGTGCTGCTGGAATTTGTTACCGCAGCAGGTATTACCTTAGGTATGGATGAACTGTATAAAGTCGACGCAGAAGCAGCAGCAAAAGAAGCCGCTGCCAAAGAAGCGGCAGCGAAAGAGGCAGCCGCAAAAGCACTGGAAGCCGAGGCTGCGGCTAAAGAGGCTGCTGCAAAAGAAGCAGCCGCTAAAGAAGCTGCGGCTAAGGCACCGGCAGCAAAACGTGTTAAACTGGACGGTGGTGGTGGTAGCACCGGTATGGACAAGAAATACAGCATCGGTTTGGATATTGGCACGAATAGCGTGGGTTGGGCCGTTATTACCGACGAGTACAAAGTGCCGTCCAAGAAATTCAAAGTGCTGGGCAATACCGATCGCCATAGCATCAAGAAAAATCTGATTGGCGCACTGCTGTTCGACAGCGGTGAGACTGCCGAAGCTACGCGTCTGAAGCGTACGGCGCGTCGTCGCTACACCCGCCGTAAGAACCGTATTTGCTATCTGCAAGAAATCTTCAGCAACGAAATGGCCAAAGTTGATGATAGCTTTTTTCACCGCCTGGAAGAGAGCTTTCTGGTGGAAGAGGATAAGAAACACGAGCGCCATCCGATTTTTGGTAACATTGTCGATGAAGTGGCATACCATGAGAAGTACCCGACCATCTACCACCTTCGTAAGAAACTGGTGGACAGCACCGATAAAGCTGATCTGCGTCTGATTTACCTGGCGCTGGCCCACATGATTAAGTTTCGCGGTCATTTTCTGATCGAGGGCGATCTGAATCCGGACAATTCTGATGTTGACAAGCTGTTTATTCAACTTGTACAGACCTACAACCAGTTGTTCGAAGAGAACCCGATCAATGCGAGCGGTGTTGATGCCAAAGCAATTCTGAGCGCACGCCTGAGCAAATCTCGCCGTTTGGAGAACCTGATTGCACAGCTGCCGGGTGAGAAGAAAAACGGTCTGTTCGGCAATCTGATTGCACTGTCCCTGGGCTTGACCCCGAATTTTAAGAGCAACTTCGACCTGGCCGAAGATGCGAAGCTCCAATTGAGCAAAGACACCTACGACGATGACCTGGACAATCTGCTGGCCCAGATTGGCGACCAGTACGCAGATCTGTTCTTGGCTGCGAAAAACCTGAGCGATGCAATTCTGCTGTCGGACATCCTGCGCGTGAATACGGAAATCACGAAAGCGCCTCTGAGCGCGTCTATGATCAAGCGCTATGACGAGCACCACCAAGATCTGACCCTGCTGAAAGCTCTGGTGAGACAACAATTGCCAGAGAAGTATAAAGAAATTTTCTTTGACCAGAGCAAAAACGGCTATGCGGGTTACATTGACGGTGGCGCCAGCCAAGAAGAGTTCTACAAATTCATTAAGCCTATCCTGGAGAAAATGGATGGCACCGAAGAACTGCTGGTAAAGCTGAATCGTGAAGATCTGCTGCGCAAACAGCGCACTTTTGATAACGGTAGCATTCCGCACCAGATCCATCTGGGTGAGTTGCACGCGATTTTGCGTCGCCAGGAAGATTTTTATCCGTTCTTGAAAGACAACCGTGAGAAAATCGAGAAAATTCTGACGTTCCGTATCCCGTATTATGTCGGCCCGCTGGCGCGTGGTAATAGCCGCTTCGCGTGGATGACCCGCAAATCAGAGGAAACGATTACCCCGTGGAATTTTGAGGAAGTTGTTGATAAGGGTGCAAGCGCGCAGTCGTTCATTGAGCGTATGACCAACTTTGACAAGAATTTGCCGAATGAAAAAGTCTTGCCGAAGCACTCTCTGCTGTACGAGTATTTTACCGTTTACAACGAATTGACCAAGGTTAAATACGTCACCGAAGGCATGCGCAAACCGGCCTTCCTGAGCGGCGAGCAGAAAAAAGCAATCGTTGACCTCTTGTTTAAGACCAACCGCAAGGTTACGGTCAAACAACTGAAAGAGGACTATTTCAAGAAAATTGAATGTTTTGACTCCGTAGAGATCTCCGGTGTTGAGGACCGTTTCAACGCGAGCCTGGGCACCTACCATGATCTGCTGAAAATTATTAAAGACAAAGATTTTCTGGACAACGAAGAGAACGAAGATATTCTGGAAGATATCGTTCTGACCCTGACGCTGTTCGAAGATCGTGAGATGATTGAGGAACGTCTGAAAACCTACGCACACTTGTTCGATGACAAAGTTATGAAACAGCTGAAGCGTCGTCGTTACACAGGTTGGGGCCGTCTGAGCCGTAAGCTTATCAATGGTATCCGTGACAAACAGAGCGGTAAGACGATTCTGGACTTTCTGAAGTCAGATGGCTTCGCCAATCGCAACTTTATGCAACTGATTCATGACGACTCTCTGACGTTCAAGGAAGATATCCAAAAGGCACAGGTGAGCGGTCAGGGTGATAGCCTGCATGAGCATATCGCGAACCTGGCGGGTAGCCCGGCTATCAAAAAGGGTATCTTACAGACTGTGAAAGTTGTGGATGAATTGGTTAAGGTTATGGGTCGTCACAAACCGGAAAATATTGTGATCGAGATGGCACGTGAAAATCAGACGACGCAAAAGGGTCAAAAAAATTCTCGTGAGCGCATGAAACGTATTGAAGAGGGTATCAAAGAATTGGGCAGCCAAATTCTGAAAGAACACCCGGTCGAGAACACCCAGCTGCAAAACGAAAAACTGTATTTATACTATCTGCAGAACGGTCGTGACATGTACGTGGATCAAGAACTGGACATCAATCGTTTGAGCGATTACGATGTTGATCATATTGTGCCTCAGAGCTTTCTGAAAGACGATTCGATCGACAACAAAGTGCTGACCCGTAGCGACAAGAATCGTGGTAAGAGCGATAACGTGCCGAGCGAAGAAGTCGTTAAGAAAATGAAAAACTACTGGCGTCAGCTGCTGAACGCCAAGCTGATTACCCAGCGTAAGTTCGATAACCTGACGAAAGCCGAGCGTGGAGGCCTGAGCGAGCTGGACAAGGCCGGCTTTATCAAGCGTCAACTGGTGGAAACCCGTCAGATCACTAAACATGTGGCACAGATCCTGGACTCCCGCATGAATACGAAATATGACGAGAATGACAAGTTGATCCGTGAAGTCAAAGTTATTACGCTGAAAAGCAAACTGGTGTCCGATTTCCGTAAAGACTTCCAGTTCTATAAAGTCCGTGAAATCAACAACTATCATCACGCCCACGATGCGTACTTGAACGCTGTTGTGGGCACCGCACTGATCAAGAAATACCCTAAGCTCGAAAGCGAGTTTGTCTATGGTGACTATAAAGTTTACGACGTGCGTAAGATGATCGCCAAGAGCGAGCAAGAAATTGGTAAGGCTACCGCAAAGTACTTTTTCTACAGCAACATCATGAACTTCTTCAAAACCGAGATTACCCTGGCGAACGGTGAGATCCGTAAACGGCCGCTGATTGAGACTAATGGCGAAACGGGCGAGATTGTGTGGGACAAGGGTCGCGATTTCGCTACGGTTCGTAAGGTCCTGAGCATGCCGCAAGTTAACATTGTCAAGAAAACTGAAGTGCAGACGGGTGGCTTTAGCAAAGAATCCATCCTGCCGAAGCGTAATAGCGATAAACTTATCGCGCGTAAAAAAGACTGGGACCCAAAGAAATATGGCGGCTTTGATAGCCCGACCGTCGCGTATAGCGTGTTAGTGGTCGCGAAAGTTGAAAAGGGCAAGAGCAAGAAACTGAAGTCCGTCAAAGAACTTCTGGGTATCACCATCATGGAACGTAGCTCCTTTGAGAAGAACCCGATTGACTTCTTAGAGGCGAAGGGTTATAAAGAAGTCAAAAAAGACCTGATTATCAAGCTGCCGAAGTACAGCCTGTTTGAGTTGGAGAATGGTCGTAAGCGCATGCTGGCGAGCGCGGGTGAGCTGCAAAAGGGCAACGAACTGGCGCTGCCGTCGAAATACGTCAATTTTCTGTACCTGGCCAGCCACTACGAAAAGCTGAAGGGTTCTCCGGAAGATAACGAACAAAAGCAACTGTTCGTTGAGCAACATAAACACTACTTGGACGAAATCATCGAGCAAATTAGCGAATTTAGCAAACGTGTCATCCTGGCGGACGCGAATCTGGACAAGGTCCTGTCTGCATACAATAAGCATCGCGACAAACCAATTCGTGAGCAAGCGGAGAATATCATCCACCTGTTTACGCTGACCAACCTAGGTGCGCCGGCGGCATTCAAGTATTTCGATACGACCATCGACCGCAAGCGCTATACCAGCACCAAAGAGGTCCTGGACGCGACCCTGATCCACCAGAGCATTACCGGCTTATACGAAACCCGTATTGATTTGAGCCAACTGGGTGGCGATGAATTCCCGAAAAAAAAGCGCAAAGTTGGTGGCGGTGGTAGCCCGAAAAAGAAACGTAAAGTG Human codon optimized RFP-SpCas9 DNA sequence(SEQ ID NO: 63) ATGGTTAGCAAAGGTGAAGCCGTGATTAAAGAATTTATGCGCTTTAAGGTTCACATGGAAGGTAGCATGAATGGCCATGAATTTGAAATTGAAGGTGAAGGCGAAGGTCGTCCGTATGAAGGCACCCAGACCGCAAAACTGAAAGTTACCAAAGGTGGTCCGCTGCCGTTTAGCTGGGATATTCTGAGTCCGCAGTTTATGTATGGTAGCCGTGCATTTACCAAACATCCGGCAGATATTCCGGATTATTACAAACAGAGCTTTCCGGAAGGTTTTAAATGGGAACGTGTGATGAATTTTGAAGATGGTGGTGCAGTTACCGTTACACAGGATACCAGCCTGGAAGATGGCACCCTGATCTATAAAGTTAAACTGCGTGGCACCAATTTTCCGCCTGATGGTCCGGTTATGCAGAAAAAAACAATGGGTTGGGAAGCAAGCACCGAACGTCTGTATCCTGAAGATGGCGTTCTGAAAGGTGATATCAAAATGGCACTGCGTCTGAAAGATGGCGGTCGTTATCTGGCAGATTTCAAAACCACCTATAAAGCCAAAAAACCTGTTCAGATGCCTGGTGCCTATAATGTTGATCGTAAACTGGATATTACCAGCCACAACGAAGATTATACCGTTGTGGAACAGTATGAACGTAGCGAAGGCCGTCATAGCACAGGTGGTATGGATGAACTGTATAAAGTCGACAGCGGTGGTAGCAGCGGTGGTTCAAGCGGTAGCGAAACACCGGGTACAAGCGAAAGCGCAACACCGGAAAGCAGTGGTGGTAGTTCAGGTGGTAGTCCGGCAGCAAAACGTGTGAAACTGGATGGCGGTGGCGGTAGCACCGGTATGGACAAGAAATACAGCATCGGTTTGGATATTGGCACGAATAGCGTGGGTTGGGCCGTTATTACCGACGAGTACAAAGTGCCGTCCAAGAAATTCAAAGTGCTGGGCAATACCGATCGCCATAGCATCAAGAAAAATCTGATTGGCGCACTGCTGTTCGACAGCGGTGAGACTGCCGAAGCTACGCGTCTGAAGCGTACGGCGCGTCGTCGCTACACCCGCCGTAAGAACCGTATTTGCTATCTGCAAGAAATCTTCAGCAACGAAATGGCCAAAGTTGATGATAGCTTTTTTCACCGCCTGGAAGAGAGCTTTCTGGTGGAAGAGGATAAGAAACACGAGCGCCATCCGATTTTTGGTAACATTGTCGATGAAGTGGCATACCATGAGAAGTACCCGACCATCTACCACCTTCGTAAGAAACTGGTGGACAGCACCGATAAAGCTGATCTGCGTCTGATTTACCTGGCGCTGGCCCACATGATTAAGTTTCGCGGTCATTTTCTGATCGAGGGCGATCTGAATCCGGACAATTCTGATGTTGACAAGCTGTTTATTCAACTTGTACAGACCTACAACCAGTTGTTCGAAGAGAACCCGATCAATGCGAGCGGTGTTGATGCCAAAGCAATTCTGAGCGCACGCCTGAGCAAATCTCGCCGTTTGGAGAACCTGATTGCACAGCTGCCGGGTGAGAAGAAAAACGGTCTGTTCGGCAATCTGATTGCACTGTCCCTGGGCTTGACCCCGAATTTTAAGAGCAACTTCGACCTGGCCGAAGATGCGAAGCTCCAATTGAGCAAAGACACCTACGACGATGACCTGGACAATCTGCTGGCCCAGATTGGCGACCAGTACGCAGATCTGTTCTTGGCTGCGAAAAACCTGAGCGATGCAATTCTGCTGTCGGACATCCTGCGCGTGAATACGGAAATCACGAAAGCGCCTCTGAGCGCGTCTATGATCAAGCGCTATGACGAGCACCACCAAGATCTGACCCTGCTGAAAGCTCTGGTGAGACAACAATTGCCAGAGAAGTATAAAGAAATTTTCTTTGACCAGAGCAAAAACGGCTATGCGGGTTACATTGACGGTGGCGCCAGCCAAGAAGAGTTCTACAAATTCATTAAGCCTATCCTGGAGAAAATGGATGGCACCGAAGAACTGCTGGTAAAGCTGAATCGTGAAGATCTGCTGCGCAAACAGCGCACTTTTGATAACGGTAGCATTCCGCACCAGATCCATCTGGGTGAGTTGCACGCGATTTTGCGTCGCCAGGAAGATTTTTATCCGTTCTTGAAAGACAACCGTGAGAAAATCGAGAAAATTCTGACGTTCCGTATCCCGTATTATGTCGGCCCGCTGGCGCGTGGTAATAGCCGCTTCGCGTGGATGACCCGCAAATCAGAGGAAACGATTACCCCGTGGAATTTTGAGGAAGTTGTTGATAAGGGTGCAAGCGCGCAGTCGTTCATTGAGCGTATGACCAACTTTGACAAGAATTTGCCGAATGAAAAAGTCTTGCCGAAGCACTCTCTGCTGTACGAGTATTTTACCGTTTACAACGAATTGACCAAGGTTAAATACGTCACCGAAGGCATGCGCAAACCGGCCTTCCTGAGCGGCGAGCAGAAAAAAGCAATCGTTGACCTCTTGTTTAAGACCAACCGCAAGGTTACGGTCAAACAACTGAAAGAGGACTATTTCAAGAAAATTGAATGTTTTGACTCCGTAGAGATCTCCGGTGTTGAGGACCGTTTCAACGCGAGCCTGGGCACCTACCATGATCTGCTGAAAATTATTAAAGACAAAGATTTTCTGGACAACGAAGAGAACGAAGATATTCTGGAAGATATCGTTCTGACCCTGACGCTGTTCGAAGATCGTGAGATGATTGAGGAACGTCTGAAAACCTACGCACACTTGTTCGATGACAAAGTTATGAAACAGCTGAAGCGTCGTCGTTACACAGGTTGGGGCCGTCTGAGCCGTAAGCTTATCAATGGTATCCGTGACAAACAGAGCGGTAAGACGATTCTGGACTTTCTGAAGTCAGATGGCTTCGCCAATCGCAACTTTATGCAACTGATTCATGACGACTCTCTGACGTTCAAGGAAGATATCCAAAAGGCACAGGTGAGCGGTCAGGGTGATAGCCTGCATGAGCATATCGCGAACCTGGCGGGTAGCCCGGCTATCAAAAAGGGTATCTTACAGACTGTGAAAGTTGTGGATGAATTGGTTAAGGTTATGGGTCGTCACAAACCGGAAAATATTGTGATCGAGATGGCACGTGAAAATCAGACGACGCAAAAGGGTCAAAAAAATTCTCGTGAGCGCATGAAACGTATTGAAGAGGGTATCAAAGAATTGGGCAGCCAAATTCTGAAAGAACACCCGGTCGAGAACACCCAGCTGCAAAACGAAAAACTGTATTTATACTATCTGCAGAACGGTCGTGACATGTACGTGGATCAAGAACTGGACATCAATCGTTTGAGCGATTACGATGTTGATCATATTGTGCCTCAGAGCTTTCTGAAAGACGATTCGATCGACAACAAAGTGCTGACCCGTAGCGACAAGAATCGTGGTAAGAGCGATAACGTGCCGAGCGAAGAAGTCGTTAAGAAAATGAAAAACTACTGGCGTCAGCTGCTGAACGCCAAGCTGATTACCCAGCGTAAGTTCGATAACCTGACGAAAGCCGAGCGTGGAGGCCTGAGCGAGCTGGACAAGGCCGGCTTTATCAAGCGTCAACTGGTGGAAACCCGTCAGATCACTAAACATGTGGCACAGATCCTGGACTCCCGCATGAATACGAAATATGACGAGAATGACAAGTTGATCCGTGAAGTCAAAGTTATTACGCTGAAAAGCAAACTGGTGTCCGATTTCCGTAAAGACTTCCAGTTCTATAAAGTCCGTGAAATCAACAACTATCATCACGCCCACGATGCGTACTTGAACGCTGTTGTGGGCACCGCACTGATCAAGAAATACCCTAAGCTCGAAAGCGAGTTTGTCTATGGTGACTATAAAGTTTACGACGTGCGTAAGATGATCGCCAAGAGCGAGCAAGAAATTGGTAAGGCTACCGCAAAGTACTTTTTCTACAGCAACATCATGAACTTCTTCAAAACCGAGATTACCCTGGCGAACGGTGAGATCCGTAAACGGCCGCTGATTGAGACTAATGGCGAAACGGGCGAGATTGTGTGGGACAAGGGTCGCGATTTCGCTACGGTTCGTAAGGTCCTGAGCATGCCGCAAGTTAACATTGTCAAGAAAACTGAAGTGCAGACGGGTGGCTTTAGCAAAGAATCCATCCTGCCGAAGCGTAATAGCGATAAACTTATCGCGCGTAAAAAAGACTGGGACCCAAAGAAATATGGCGGCTTTGATAGCCCGACCGTCGCGTATAGCGTGTTAGTGGTCGCGAAAGTTGAAAAGGGCAAGAGCAAGAAACTGAAGTCCGTCAAAGAACTTCTGGGTATCACCATCATGGAACGTAGCTCCTTTGAGAAGAACCCGATTGACTTCTTAGAGGCGAAGGGTTATAAAGAAGTCAAAAAAGACCTGATTATCAAGCTGCCGAAGTACAGCCTGTTTGAGTTGGAGAATGGTCGTAAGCGCATGCTGGCGAGCGCGGGTGAGCTGCAAAAGGGCAACGAACTGGCGCTGCCGTCGAAATACGTCAATTTTCTGTACCTGGCCAGCCACTACGAAAAGCTGAAGGGTTCTCCGGAAGATAACGAACAAAAGCAACTGTTCGTTGAGCAACATAAACACTACTTGGACGAAATCATCGAGCAAATTAGCGAATTTAGCAAACGTGTCATCCTGGCGGACGCGAATCTGGACAAGGTCCTGTCTGCATACAATAAGCATCGCGACAAACCAATTCGTGAGCAAGCGGAGAATATCATCCACCTGTTTACGCTGACCAACCTAGGTGCGCCGGCGGCATTCAAGTATTTCGATACGACCATCGACCGCAAGCGCTATACCAGCACCAAAGAGGTCCTGGACGCGACCCTGATCCACCAGAGCATTACCGGCTTATACGAAACCCGTATTGATTTGAGCCAACTGGGTGGCGATGAATTCCCGAAAAAAAAGCGCAAAGTTGGTGGCGGTGGTAGCCCGAAAAA GAAACGTAAAGTG Human codon optimized GFP-eSpCas9 DNA sequence (SEQ ID NO: 64)ATGGTTAGCAAAGGTGAAGAACTGTTTACAGGTGTTGTTCCGATTCTGGTTGAACTGGATGGTGATGTTAATGGCCACAAATTTTCAGTTAGCGGTGAAGGCGAAGGTGATGCAACCTATGGTAAACTGACCCTGAAATTTATCTGTACCACCGGCAAACTGCCGGTTCCGTGGCCGACACTGGTTACCACACTGACCTATGGTGTTCAGTGTTTTAGCCGTTATCCGGATCACATGAAACAGCACGATTTTTTCAAAAGCGCAATGCCGGAAGGTTATGTTCAAGAACGTACCATCTTCTTCAAAGATGACGGCAACTATAAAACCCGTGCCGAAGTTAAATTTGAAGGTGATACCCTGGTGAATCGCATTGAACTGAAAGGCATCGATTTTAAAGAGGATGGTAATATCCTGGGCCACAAACTGGAATATAATTATAATAGCCACAACGTGTACATCATGGCCGACAAACAGAAAAATGGCATCAAAGTGAACTTCAAGATCCGCCATAATATTGAAGATGGTTCAGTTCAGCTGGCCGATCATTATCAGCAGAATACCCCGATTGGTGATGGTCCGGTTCTGCTGCCGGATAATCATTATCTGAGCACCCAGAGCAAACTGAGCAAAGATCCGAATGAAAAACGTGATCACATGGTGCTGCTGGAATTTGTTACCGCAGCAGGTATTACCTTAGGTATGGATGAACTGTATAAAGTCGACAGCGGTGGTAGCAGCGGTGGTTCAAGCGGTAGCGAAACACCGGGTACAAGCGAAAGCGCAACACCGGAAAGCAGTGGTGGTAGCTCAGGTGGTAGTCCGGCAGCAAAACGTGTTAAACTGGACGGTGGTGGTGGTAGCACCGGTATGGACAAGAAATACAGCATCGGTTTGGATATTGGCACGAATAGCGTGGGTTGGGCCGTTATTACCGACGAGTACAAAGTGCCGTCCAAGAAATTCAAAGTGCTGGGCAATACCGATCGCCATAGCATCAAGAAAAATCTGATTGGCGCACTGCTGTTCGACAGCGGTGAGACTGCCGAAGCTACGCGTCTGAAGCGTACGGCGCGTCGTCGCTACACCCGCCGTAAGAACCGTATTTGCTATCTGCAAGAAATCTTCAGCAACGAAATGGCCAAAGTTGATGATAGCTTTTTTCACCGCCTGGAAGAGAGCTTTCTGGTGGAAGAGGATAAGAAACACGAGCGCCATCCGATTTTTGGTAACATTGTCGATGAAGTGGCATACCATGAGAAGTACCCGACCATCTACCACCTTCGTAAGAAACTGGTGGACAGCACCGATAAAGCTGATCTGCGTCTGATTTACCTGGCGCTGGCCCACATGATTAAGTTTCGCGGTCATTTTCTGATCGAGGGCGATCTGAATCCGGACAATTCTGATGTTGACAAGCTGTTTATTCAACTTGTACAGACCTACAACCAGTTGTTCGAAGAGAACCCGATCAATGCGAGCGGTGTTGATGCCAAAGCAATTCTGAGCGCACGCCTGAGCAAATCTCGCCGTTTGGAGAACCTGATTGCACAGCTGCCGGGTGAGAAGAAAAACGGTCTGTTCGGCAATCTGATTGCACTGTCCCTGGGCTTGACCCCGAATTTTAAGAGCAACTTCGACCTGGCCGAAGATGCGAAGCTCCAATTGAGCAAAGACACCTACGACGATGACCTGGACAATCTGCTGGCCCAGATTGGCGACCAGTACGCAGATCTGTTCTTGGCTGCGAAAAACCTGAGCGATGCAATTCTGCTGTCGGACATCCTGCGCGTGAATACGGAAATCACGAAAGCGCCTCTGAGCGCGTCTATGATCAAGCGCTATGACGAGCACCACCAAGATCTGACCCTGCTGAAAGCTCTGGTGAGACAACAATTGCCAGAGAAGTATAAAGAAATTTTCTTTGACCAGAGCAAAAACGGCTATGCGGGTTACATTGACGGTGGCGCCAGCCAAGAAGAGTTCTACAAATTCATTAAGCCTATCCTGGAGAAAATGGATGGCACCGAAGAACTGCTGGTAAAGCTGAATCGTGAAGATCTGCTGCGCAAACAGCGCACTTTTGATAACGGTAGCATTCCGCACCAGATCCATCTGGGTGAGTTGCACGCGATTTTGCGTCGCCAGGAAGATTTTTATCCGTTCTTGAAAGACAACCGTGAGAAAATCGAGAAAATTCTGACGTTCCGTATCCCGTATTATGTCGGCCCGCTGGCGCGTGGTAATAGCCGCTTCGCGTGGATGACCCGCAAATCAGAGGAAACGATTACCCCGTGGAATTTTGAGGAAGTTGTTGATAAGGGTGCAAGCGCGCAGTCGTTCATTGAGCGTATGACCAACTTTGACAAGAATTTGCCGAATGAAAAAGTCTTGCCGAAGCACTCTCTGCTGTACGAGTATTTTACCGTTTACAACGAATTGACCAAGGTTAAATACGTCACCGAAGGCATGCGCAAACCGGCCTTCCTGAGCGGCGAGCAGAAAAAAGCAATCGTTGACCTCTTGTTTAAGACCAACCGCAAGGTTACGGTCAAACAACTGAAAGAGGACTATTTCAAGAAAATTGAATGTTTTGACTCCGTAGAGATCTCCGGTGTTGAGGACCGTTTCAACGCGAGCCTGGGCACCTACCATGATCTGCTGAAAATTATTAAAGACAAAGATTTTCTGGACAACGAAGAGAACGAAGATATTCTGGAAGATATCGTTCTGACCCTGACGCTGTTCGAAGATCGTGAGATGATTGAGGAACGTCTGAAAACCTACGCACACTTGTTCGATGACAAAGTTATGAAACAGCTGAAGCGTCGTCGTTACACAGGTTGGGGCCGTCTGAGCCGTAAGCTTATCAATGGTATCCGTGACAAACAGAGCGGTAAGACGATTCTGGACTTTCTGAAGTCAGATGGCTTCGCCAATCGCAACTTTATGCAACTGATTCATGACGACTCTCTGACGTTCAAGGAAGATATCCAAAAGGCACAGGTGAGCGGTCAGGGTGATAGCCTGCATGAGCATATCGCGAACCTGGCGGGTAGCCCGGCTATCAAAAAGGGTATCTTACAGACTGTGAAAGTTGTGGATGAATTGGTTAAGGTTATGGGTCGTCACAAACCGGAAAATATTGTGATCGAGATGGCACGTGAAAATCAGACGACGCAAAAGGGTCAAAAAAATTCTCGTGAGCGCATGAAACGTATTGAAGAGGGTATCAAAGAATTGGGCAGCCAAATTCTGAAAGAACACCCGGTCGAGAACACCCAGCTGCAAAACGAAAAACTGTATTTATACTATCTGCAGAACGGTCGTGACATGTACGTGGATCAAGAACTGGACATCAATCGTTTGAGCGATTACGATGTTGATCATATTGTGCCTCAGAGCTTTCTGGCGGACGATTCGATCGACAACAAAGTGCTGACCCGTAGCGACAAGAATCGTGGTAAGAGCGATAACGTGCCGAGCGAAGAAGTCGTTAAGAAAATGAAAAACTACTGGCGTCAGCTGCTGAACGCCAAGCTGATTACCCAGCGTAAGTTCGATAACCTGACGAAAGCCGAGCGTGGAGGCCTGAGCGAGCTGGACAAGGCCGGCTTTATCAAGCGTCAACTGGTGGAAACCCGTCAGATCACTAAACATGTGGCACAGATCCTGGACTCCCGCATGAATACGAAATATGACGAGAATGACAAGTTGATCCGTGAAGTCAAAGTTATTACGCTGAAAAGCAAACTGGTGTCCGATTTCCGTAAAGACTTCCAGTTCTATAAAGTCCGTGAAATCAACAACTATCATCACGCCCACGATGCGTACTTGAACGCTGTTGTGGGCACCGCACTGATCAAGAAATACCCTGCACTCGAAAGCGAGTTTGTCTATGGTGACTATAAAGTTTACGACGTGCGTAAGATGATCGCCAAGAGCGAGCAAGAAATTGGTAAGGCTACCGCAAAGTACTTTTTCTACAGCAACATCATGAACTTCTTCAAAACCGAGATTACCCTGGCGAACGGTGAGATCCGTAAAGCGCCGCTGATTGAGACTAATGGCGAAACGGGCGAGATTGTGTGGGACAAGGGTCGCGATTTCGCTACGGTTCGTAAGGTCCTGAGCATGCCGCAAGTTAACATTGTCAAGAAAACTGAAGTGCAGACGGGTGGCTTTAGCAAAGAATCCATCCTGCCGAAGCGTAATAGCGATAAACTTATCGCGCGTAAAAAAGACTGGGACCCAAAGAAATATGGCGGCTTTGATAGCCCGACCGTCGCGTATAGCGTGTTAGTGGTCGCGAAAGTTGAAAAGGGCAAGAGCAAGAAACTGAAGTCCGTCAAAGAACTTCTGGGTATCACCATCATGGAACGTAGCTCCTTTGAGAAGAACCCGATTGACTTCTTAGAGGCGAAGGGTTATAAAGAAGTCAAAAAAGACCTGATTATCAAGCTGCCGAAGTACAGCCTGTTTGAGTTGGAGAATGGTCGTAAGCGCATGCTGGCGAGCGCGGGTGAGCTGCAAAAGGGCAACGAACTGGCGCTGCCGTCGAAATACGTCAATTTTCTGTACCTGGCCAGCCACTACGAAAAGCTGAAGGGTTCTCCGGAAGATAACGAACAAAAGCAACTGTTCGTTGAGCAACATAAACACTACTTGGACGAAATCATCGAGCAAATTAGCGAATTTAGCAAACGTGTCATCCTGGCGGACGCGAATCTGGACAAGGTCCTGTCTGCATACAATAAGCATCGCGACAAACCAATTCGTGAGCAAGCGGAGAATATCATCCACCTGTTTACGCTGACCAACCTAGGTGCGCCGGCGGCATTCAAGTATTTCGATACGACCATCGACCGCAAGCGCTATACCAGCACCAAAGAGGTCCTGGACGCGACCCTGATCCACCAGAGCATTACCGGCTTATACGAAACCCGTATTGATTTGAGCCAACTGGGTGGCGATGAATTCCCGAAAAAAAAGCGCAAAGTTGGTGGCGGTGGTAGCCCGAAAAAGAAACGTAAAGTG 

Example 2: Editing Efficiency Comparison with Commercial Products

Two commercial GFP-SpCas9 fusion protein products, GenCrisprNLS-Cas9-EGFP Nuclease and ArciTect Cas9-eGFP Nuclease, were purchasedfrom GenScript (Piscataway, N.J.) and Stemcell Technologies (Vancouver,Canada), respectively. Human U2OS cells (0.2×10⁶) and HEK293 cells(0.3×10⁶) were transfected with 50 pmol of GenCrispr NLS-Cas9-EGFPNuclease, or ArciTect Cas9-eGFP Nuclease, or the GFP-SpCas9 protein ofthe current invention, in combination with 150 pmol each of fourchemically synthesized sgRNAs targeting the Human EMX1, HEKSite4,VEGFA3, HPRT loci. The guide sequences are: 5′-GAGUCCGAGCAGAAGAAGAA-3′(EMX1) (SEQ ID NO:51), 5′-GGCACUGCGGCUGGAGGUGG-3′ (HEKSite4) (SEQ IDNO:52), 5′GGUGAGUGAGUGUGUGCGUG-3′ (VEGFA3) (SEQ ID NO: 66), and5′-GGUCACUUUUAACACACCCA-3′ (HPRT) (SEQ ID NO:53). Transfection wascarried out using Nucleofection Solution V and an Amaxa instrument.Cells were maintained at 37° C. and 5% CO₂ for three days beforeharvested for gene editing analysis. Genomic DNA was prepared usingQuickExtract DNA extraction solution. Each targeted genomic region wasPCR amplified using a pair of primers consisting of target-specificsequences and next generation sequencing (NGS) adaptors. The primers arelisted in the following table:

NGS primer sequences Target Primer sequence (5′-3′) EMX1 Forward:TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGNNNNNNCC CCAGTGGCTGCTCT (SEQ ID NO: 54)Reverse: GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGNNNNNNCCAGGCCTCCCCAAAGC (SEQ ID NO: 55) HEKSite4 Forward:TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGNNNNNNGGAACCCAGGTAGCCAGAGA (SEQ ID NO: 56) Reverse:GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGNNNNNNGGGGTGGGGTCAGACGT (SEQ ID NO: 57) VEGFA3 Forward:TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGNNNNNNGCCCATTCCCTCTTTAGCCA (SEQ ID NO: 58) Reverse:GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGNNNNNNGGAGCAGGAAAGTGAGGTTAC (SEQ ID NO: 59) HPRT Forward:TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGNNNNNNAATGGACACATGGGTAGTCAGG (SEQ ID NO: 60) Reverse:GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGNNNNNNGGCTTATATCCAACACTTCGTGGG (SEQ ID NO: 61)

PCR amplicons were analyzed by NGS using the Illumina MiSeq to determinethe editing efficiency of each Cas9 protein. The results in FIG. 2A andFIG. 2B show that the editing efficiencies by the GFP-SpCas9 protein ofthe current invention were several-fold higher than that of thecommercial proteins in all targets.

What is claimed is:
 1. A system comprising a fusion protein and anengineered guide RNA, wherein the fusion protein comprises aStreptococcus pyogenes Cas9 protein; a marker protein; at least onefirst linker between the Cas9 protein and the marker protein, whereinthe at least one first linker comprises SEQ ID NO:35 or SEQ ID NO:36;and a first heterologous domain that is a nuclear localization signal;wherein the fusion protein is a nuclease and cleaves both strands of adouble-stranded sequence, is a nickase and cleaves one strand of adouble-stranded sequence, or has no nuclease or nickase activity.
 2. Thesystem of claim 1, wherein the engineered guide RNA is a singlemolecule.
 3. The system of claim 1, wherein the engineered guide RNA istwo separate molecules.
 4. The system of claim 1, wherein the engineeredguide RNA sequence is optimized to facilitate base-paring within theengineered guide RNA, minimize base-paring within the engineered guideRNA, increase stability of the engineered guide RNA, facilitatetranscription of the engineered guide RNA in a eukaryotic cell, or acombination thereof.
 5. The system of claim 1, wherein the markerprotein is at the N-terminus of the fusion protein.
 6. The system ofclaim 1, wherein the fusion protein further comprises an optional secondlinker, wherein the nuclear localization signal, the marker protein, thefirst linker, the optional second linker (if present), and the Cas9protein are arranged in the following order (N-terminus to C-terminus):marker protein-first linker-nuclear localization signal-Cas9 protein;marker protein-nuclear localization signal-first linker-Cas9 protein;nuclear localization signal-marker protein-first linker-Cas9 protein;marker protein-first linker-nuclear localization signal-firstlinker-Cas9 protein; or nuclear localization signal-second linker-markerprotein-first linker-Cas9 protein.
 7. The system of claim 6, wherein thenuclear localization signal, the marker protein, the first linker, andthe Cas9 protein are arranged in the following order (N-terminus toC-terminus): marker protein-first linker-nuclear localizationsignal-Cas9 protein.
 8. The system of claim 1, wherein the fusionprotein further comprises at least one additional heterologous domainother than the first heterologous domain that is a nuclear localizationsignal, wherein the at least one additional heterologous domain is acell-penetrating domain, a chromatin modulating motif, an epigeneticmodification domain, a transcriptional regulation domain, an RNA aptamerbinding domain, or combination thereof.
 9. The system of claim 1,wherein the first heterologous domain that is a nuclear localizationsignal comprises the sequence PAAKRVKLD (SEQ ID NO:6).
 10. The system ofclaim 9, wherein the fusion protein further comprises two nuclearlocalization signals comprising the sequence PKKKRKV (SEQ ID NO:1). 11.The system of claim 1, wherein the marker protein has an amino acidsequence comprising SEQ ID NO:19 or
 20. 12. The system of claim 1,wherein the marker protein has an amino acid sequence consisting of SEQID NO:19 or
 20. 13. The system of claim 1, wherein the fusion proteinhas an amino acid sequence having at least 95% sequence identity withSEQ ID NOS:48, 49, or
 50. 14. The system of claim 1, wherein the fusionprotein has an amino acid sequence having at least 99% sequence identitywith SEQ ID NOS:48, 49, or
 50. 15. The system of claim 1, wherein thefusion protein has an amino acid sequence of SEQ ID NOS:48, 49, or 50.16. A plurality of nucleic acids encoding the system of claim 1, theplurality of nucleic acids comprising at least one nucleic acid encodingthe fusion protein, and at least one nucleic acid encoding theengineered guide RNA.
 17. The plurality of nucleic acids of claim 16,wherein the at least one nucleic acid encoding the fusion protein isRNA.
 18. The plurality of nucleic acids of claim 16, wherein the atleast one nucleic acid encoding the fusion protein is DNA.
 19. Theplurality of nucleic acids of claim 16, wherein the at least one nucleicacid encoding the fusion protein is codon optimized for expression in aeukaryotic cell.
 20. The plurality of nucleic acids of claim 19, whereinthe eukaryotic cell is a human cell, a non-human mammalian cell, anon-mammalian vertebrate cell, an invertebrate cell, a plant cell, or asingle cell eukaryotic organism.
 21. The plurality of nucleic acids ofclaim 16, wherein the at least one nucleic acid encoding the engineeredguide RNA is DNA.
 22. The plurality of nucleic acids of claim 16,wherein the at least one nucleic acid encoding the fusion protein isoperably linked to a phage promoter sequence for in vitro RNA synthesisor protein expression in a bacterial cell, and the at least one nucleicacid encoding the engineered guide RNA is operably linked to a phagepromoter sequence for in vitro RNA synthesis.
 23. The plurality ofnucleic acids of claim 16, wherein the at least one nucleic acidencoding the fusion protein is operably linked to a eukaryotic promotersequence for expression in a eukaryotic cell, and the at least onenucleic acid encoding the engineered guide RNA is operably linked to aeukaryotic promoter sequence for expression in a eukaryotic cell.
 24. Atleast one vector comprising the plurality of nucleic acids of claim 16.25. The at least one vector of claim 24, which is a plasmid vector, aviral vector, or a self-replicating viral RNA replicon.
 26. A eukaryoticcell comprising at least one system as defined in claim
 1. 27. Theeukaryotic cell of claim 26, which is a human cell, a non-humanmammalian cell, a plant cell, a non-mammalian vertebrate cell, aninvertebrate cell, or a single cell eukaryotic organism.
 28. Theeukaryotic cell of claim 26, which is in vivo, ex vivo, or in vitro. 29.A method for determining chromosome identity and location within aliving eukaryotic cell or chemically fixed eukaryotic cell, the methodcomprising introducing the system of claim 1 into the living orchemically fixed eukaryotic cell and detecting a signal from the markerprotein.
 30. The method of claim 29, wherein the eukaryotic cell is ahuman cell, a non-human mammalian cell, a plant cell, a non-mammalianvertebrate cell, an invertebrate cell, or a single cell eukaryoticorganism.